Current Developments on Chemical Compositions, Biosynthesis, Color Properties and Health Benefits of Black Goji Anthocyanins: An Updated Review

Abstract

Lycium ruthenicum is a therapeutic plant and its fruits (black goji) are commonly used as a traditional Chinese medicine. This review comprehensively discusses the recent research developments of black goji anthocyanins (BGAs), including chemical compositions, biosynthesis, color properties and health benefits. Among the 39 identified BGAs, most are 3,5-diglycoside derivatives of petunidin (>95%) with an individual anthocyanin [petunidin 3-O-rutinoside (trans-p-coumaroyl)-5-O-glucoside], accounting for 80% of the total BGAs. Due to their unique anthocyanin profile, BGAs possess various health benefits, including antioxidant activities, α-glucosidase inhibiting activity, alleviating insulin resistance, improving mitochondrial function, anti-inflammatory effects, etc., and therefore have the potential to treat a range of chronic diseases, such as type 2 diabetes mellitus, memory disorders, stroke, colitis, atherosclerosis, cardiovascular and cerebrovascular diseases. In addition, BGAs exhibit a pH-dependent “red-purple-blue” pattern of color change and thus could be used as natural colorants and to prepare smart food packaging materials. This review is valuable for broad applications of BGAs as promising natural colorants, functional foods and potential herbal medicines.

1. Introduction

Lycium ruthenicum, a therapeutic plant that belongs to the family of Solanaceae and genus of Lycium, is mainly distributed in China, Mongolia, Central Asia and North Africa, and grows under the environment of special geography (high altitude) and arid climate (low temperature, less rain and strong sunlight) [1,2]. The fruit, known as black goji, is popular in traditional Chinese medicine for disease treatment, such as heart disease, abnormal menstruation, urethral and urethral stones, tinea and furuncle, hypertension and menopause, as highlighted in the Tibetan medical classics of “Jing Zhu Ben Cao” [2,3,4,5,6]. Modern pharmacological research has demonstrated its potential use as an antioxidant, immune-enhancer, hepatic-function protector and anti-tumor, antifatigue, antiaging, anti-atherosclerotic, hypolipidemic and hypoglycemic agent [5,6,7]. The biological functions of black goji are derived from its functional components such as anthocyanins, essential oils, organic acids, trace minerals and polysaccharides [8,9,10,11]. Black goji is also popular in the food industry for preparing functional foods such as black goji juices and drinks, not only owing to its biological functions but also its attractive black-bluish color [3]. Recently, black goji consumption has remarkably increased, and the demands for functional black goji products are expanding [7,12].

Anthocyanins are abundant in black goji and they are the principal functional compounds that exhibit a wide range of health benefits (e.g., antioxidant activities, anti-cardiovascular disease and anti-tumor effects) and thus are responsible for most of black goji’s biological functions [4,7,13,14,15,16,17]. The anthocyanins are also responsible for the attractive color of the fruit [18]. To date, many researchers have explored certain aspects of black goji anthocyanins (BGAs), e.g., extraction, chemical compositions, biosynthesis and health benefits [2,7,11,14,15,19,20,21,22,23]. Gamage et al. [24] summarized the data related to the extraction, stability, health benefits and applications of BGAs. However, to our best knowledge, more information, especially on health benefits, should be given. The current dilemma is that no systematic and comprehensive information about BGAs is available, hindering its commercial application as a functional food and potential medicine. Therefore, the objective of this study was to provide an updated overview of BGAs in terms of their chemical compositions, biosynthesis, color properties and health benefits, which may promote in-depth research on this group of unique compounds and product development.

2. Chemical Compositions and Biosynthesis of BGAs

2.1. Extraction and Purification

As a class of water-soluble polyphenols, BGAs are generally extracted using an aqueous solution [25,26]. However, organic reagents including methanol, ethanol and acetone were found to be more efficient in the extraction of polyphenols [27]. Therefore, BGAs are commonly extracted using organic reagents [2,6,11,18,28] or aqueous solutions with different concentrations of organic reagents (50–80%) [1,4,14,15,16,19,20,21,22,29,30,31,32,33,34]. Ethanol is the most common reagent in BGA extraction due to its edible and safe properties [35]. The ethanol concentration, solution to black goji ratio, and extraction time show a significant effect on the extraction yield of BGAs [14]. The extractants were mostly adjusted to pH 2 or 2.5 using hydrochloric, formic or trifluoroacetic acid, and the extractions were carried out at a temperature less than 50 °C in the dark to protect BGAs from degradation.

An ultrasound-assisted system is widely adopted in BGA extraction [1,14,28,34,36,37,38], because particle collisions and cell wall disruption produced by ultrasound cavitation can promote solvent penetration into the sample matrix, enhancing the anthocyanin extraction rate [39]. The extract yield of BGAs by an optimized ultrasound-assisted extraction system, i.e., with extraction power of 348 W, extractant (90% ethanol) and material ratio of 25 mL/g, at temperature 42 °C for 29 min, was 17.92%, higher than the yield of microwave-assisted extraction and soaking extraction (16.85% and 16.34%, respectively) [36]. The efficiency of ultrasonic extraction is related to its power, as a lower extraction yield of BGAs (7.12%) was observed when the extraction power was lower, i.e., 300 W, with other parameters the same as described above [37].

Some novel techniques are also employed to enhance the efficiency of BGA extraction. A subcritical water extraction with the subcritical water flow speed of 3 mL/min at 170 °C for 55 min recovered higher amounts of BGAs (up to 26.33%) than a hot water extraction (15.76%) and a methyl alcohol extraction (21.35%) [40]. Pectinase extraction at 38 °C for 37 min using pectinase with a concentration of 52.04 mg/100 g dried black goji rendered the BGAs at 19.51 mg/g dry weight (DW) [41], which is much higher than the extraction using aqueous two-phase assisted by ultrasound (4.71 mg/g DW) [38]. β-cyclodextrin (β-CD, 1.65%) extraction with the liquid/solid ratio of 15:1 at 50 °C for 30 min produced higher extraction yields of both major [petunidin 3-O-rutinoside (trans-p-coumaroyl)-5-O-glucoside, Pt3R5G] and total BGAs than pure water, aqueous hydroxypropyl-β-CD and ethanol and methanol solutions [42]. In addition, yeast fermentation for 2 h before the extraction by 77.8% ethanol was found to increase the total BGA content by 51% in the extract [43].

Anthocyanin-rich fruits generally contain abundant other types of phenolic compounds, which complicates the qualitative and quantitative analysis [44] and influences the stability, bioactivity and color properties of anthocyanins [45]. Thus, purification is a critical factor in BGA analysis. The crude extracts of BGAs were purified by SPE C-18 column [11,28,33], YMC-Pack ODS-A column [32], Oasis MCX column [33] and macroporous resin columns including AB-8 [1,4,14,16,19,26,29,30,31,32,37], Diaion HP2 MGL [15], Amberlite XAD-7HP [6,20,30], D-101 [18,38] and XAD-6 [41]. The columns were successively rinsed with water to remove sugars and other interfering substances and then methanol or ethanol was used to elute the anthocyanin fraction. The purification technologies removed the phenolic compounds from the crude extract, reducing the complexity of the matrix. For example, MCX purification increased the purity of BGAs to 65% in contrast to 43% in crude extract and 50% in a C-18 cartridge-purified sample [33]. Particularly, XAD-7HP purification led to the purity of Pt3R5G reaching >97% [20,30].

2.2. Characterization

To date, researchers have isolated and identified 39 anthocyanins from the crude extracts or purified powder of BGAs by HPLC (e.g., HPLC-DAD and semipreparative HPLC), HPLC-MS (e.g., HPLC-DAD-ESI-MS and HPLC-ESI-ToF-MS) and NMR techniques (

Table 1. The identified anthocyanins in fresh or freeze-dried black goji ^a.

No.	Anthocyanins	Molecular Formula	References
Petundin derivatives
1	Petundin 3-O-galactoside-5-O-glucoside	C28H33O16 +	[2,6,11,28,33]
2	Petundin 3,5-O-diglucosides	C28H33O17 +	[2,6,11,18,28]
3	Petunidin 3-O-rutinoside-5-O-glucoside	C34H43O21 +	[14,19,22,33]
4	Petunidin 3-O-glucoside (maloyl)-5-O-glucoside	C31H35O19 +	[2,6,11,40]
5	Petunidin 3-O-glucoside (feruloyl)-5-O-glucoside	C38H41O20 +	[2,6,11,40]
6	Petunidin 3-O-rutinoside (feruloyl)-5-O-glucoside	C44H51O24 +	[19,22,25]
7	Petunidin 3-O-rutinoside (caffeoyl)-5-O-glucoside	C43H37O24 +	[2,6,11,19,28]
8	Petunidin 3-O-rutinoside (cis-caffeoyl)-5-O-glucoside	C43H50O24 +	[5]
9	Petunidin 3-O-rutinoside (trans-caffeoyl)-5-O-glucoside	C43H50O24 +	[5,22]
10	Petunidin 3-O-rutinoside (p-coumaroyl)-5-O-glucoside	C43H37O23 +	[14,20,22]
11	Petunidin 3-O-rutinoside (cis-p-coumaroyl)-5-O-glucoside	C43H37O23 +	[2,5,6,11,19,22,28,33]
12	Petunidin 3-O-rutinoside (trans-p-coumaroyl)-5-O-glucoside	C43H37O23 +	[1,2,5,6,11,18,19,22,28,33,42]
13	Petunidin 3-O-rutinoside (glucosyl-cis-p-coumaroyl)-5-O-glucoside	C49H53O28 +	[19,22]
14	Petunidin 3-O-rutinoside (glucosyl-trans-p-coumaroyl)-5-O-glucoside	C49H53O28 +	[19,22]
15	Petunidin 3-O-[6-O-(4-O-(cis-p-coumaroyl)-α-L-rhamnopyranosyl) -β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]	C43H49O23 +	[29,34,40]
16	Petunidin 3-O-[6-O-(4-O-(trans-p-coumaroyl)-α-L-rhamnopyranosyl) -β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]	C43H49O23 +	[29,30,32,34,40]
17	Petunidin 3-O-[6-O-(4-O-p-caffeoyl-α-L-rhamnopyranosyl) -β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]	C43H49O24 +	[14,40]
18	Petunidin 3-O-[6-O-(4-O-(trans-p-caffeoyl)-α-L-rhamnopyranosyl) -β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]	C43H49O24 +	[29,34]
19	Petunidin 3-O-[6-O-(4-O-(4-O-cis-(β-D-glucopyranoside)-p-coumaroyl) -α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]	C49H59O28 +	[14,29,34,40]
20	Petunidin3-O-[6-O-(4-O-(4-O-trans-(β-D-glucopyranoside)-p-coumaroyl) -α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]	C49H59O28 +	[14,29,34,40]
21	Petunidin 3-O-[6-O-(4-O-(4-O-(β-D-glucopyranosyl)-cis-p-coumaroyl) -α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]	C49H59O28 +	[14,40]
22	Petunidin 3-O-[6-O-(4-O-(4-O-(β-D-glucopyranosyl)-trans-p-coumaroyl) -α-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]	C49H59O28 +	[14,40]
23	Petunidin 3-O-[6-O-α-L-rhamnopyranosyl-β-D-glucopyranoside]-5-O- [β-D-glucopyranoside]	C34H43O21 +	[4]
Malvidin derivatives
24	Malvidin 3-O-rutinoside-5-O-glucoside	C35H45O21 +	[19]
25	Malvidin 3-O-rutinoside (feruloyl)-5-O-glucoside	C45H53O24 +	[19]
26	Malvidin 3-O-rutinoside (p-coumaroyl)-5-O-glucoside	C44H51O23 +	[35]
27	Malvidin 3-O-rutinoside (cis-p-coumaroyl)-5-O-glucoside	C44H51O23 +	[2,6,11,20,29,41]
28	Malvidin 3-O-rutinoside (trans-p-coumaroyl)-5-O-glucoside	C44H51O23 +	[20,22,23]
29	Malvidin 3-O-rutinoside (glucosyl-cis-p-coumaroyl)-5-O-glucoside	C50H61O28 +	[20]
30	Malvidin 3-O-rutinoside (glucosyl-trans-p-coumaroyl)-5-O-glucoside	C50H61O28 +	[20]
31	Malvidin 3-O-[6-O-(4-O-p-coumaroyl-α-L-rhamnosyl) -β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]	C44H51O23 +	[15,41]
32	Malvidin 3-O-[6-O-(4-O-(4-O-trans-(β-D-glucopyranoside)-p- coumaroyl)-a-L-rhamnopyranosyl)-β-D-glucopyranoside]-5-O- [β-D-glucopyranoside]	C44H51O23 +	[20]
Delphinidin derivatives
33	Delphinidin 3-O-rutinoside (cis-p-coumaroyl)-5-O-glucoside	C42H47O23 +	[2,6,11,20,29]
34	Delphinidin 3-O-rutinoside (trans-p-coumaroyl)-5-O-glucoside	C42H47O23 +	[2,6,11,22,29,34]
35	Delphinidin 3-O-rutinoside (glucosyl-trans-p-coumaroyl)-5-O-glucoside	C48H57O28 +	[20]
36	Delphinidin 3-O-[6-O-(4-O-p-coumaroyl-α-L-rhamnopyranosyl) -β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]	C42H47O23 +	[15,41]
37	Delphinidin 3-O-[6-O-(4-O-(trans-p-coumaroyl)-α-L-rhamnopyranosyl) -β-D-glucopyranoside]-5-O-[β-D-glucopyranoside]	C42H47O23 +	[30,35]
Peonidin derivatives
38	Peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-O-α-rhamnopyranosyl) -β-glucopyranoside]-5-O-[β-glucopyranoside]	C43H48O22 +	[27]
39	Peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-O-α-rhamnopyranosyl) -β-glucopyranoside]-5-O-[β-glucopyranoside]	C43H48O22 +	[27]

^a The anthocyanins in bold are abundant in black goji, as demonstrated by Yang et al. [22].

) [1,2,4,5,6,11,14,18,19,22,26,28,29,30,32,33,34,40]. In nature, the glycosylation of anthocyanins usually occurs at the third position in the C ring or at the third and fifth positions in the A ring (Figure 1A) through O-glycosidic bond forming mono- or di-glycosyl-anthocyanins [46], and anthocyanidin 3-O-glycosides (3-monoglylcosy-lanthocyanins) are the most common anthocyanins and twice as abundant as 3,5-O-diglycosides (3,5-diglycosyl-anthocyanins) [47]. However, as shown in Table 1, BGAs are all diglycosylated with sugar moieties, including galactoside, glucoside and rutinoside at the third and fifth positions of the aglycones, including petunidin (Pt), malvidin (Mv), delphinidin (Dp) and peonidin (Pn), and mostly monoacylated (at the sugar moieties of the third position) with acyl groups, including coumaric, caffeic, malic and ferulic acids. The existence of abundant 3,5-diglycosyl-anthocyanins is a unique feature of BGAs. This is confirmed by the reports in common berries including blue honeysuckle, grape, haskap and sweet cherry [3,48,49,50], where 3-monoglycosyl-anthocyanins are the primary anthocyanins, and 3,5-diglycosyl-anthocyanins are present only in a small proportion. The most common naturally occurring anthocyanidins are cyanidin (Cy, 50%), Dp (12%), Pn (12%), pelargonidin (Pg, 12%), Mv (7%) and Pt (7%) (Figure 1A) [47], and Pt is rarely present in berries [51]. However, Pt is the major component of black goji anthocyanidins, followed by Mv and Dp, with average contents of 5.71, 0.47 and 0.29 mg/g DW, respectively [1]. As shown in Table 1, eight Pt derivatives (petunidin 3-O-rutinoside-5-O-glucoside, petunidin 3-O-rutinoside (feruloyl)-5-O-glucoside, petunidin 3-O-rutinoside (trans-caffeoyl)-5-O-glucoside, petunidin 3-O-rutinoside (p-coumaroyl)-5-O-glucoside, petunidin 3-O-rutinoside (cis-p-coumaroyl)-5-O-glucoside, Pt3R5G, petunidin 3-O-rutinoside (glucosyl-cis-p-coumaroyl)-5-O-glucoside and petunidin 3-O-rutinoside (glucosyl-trans-p-coumaroyl)-5-O-glucoside), one Mv derivative (malvidin 3-O-rutinoside (trans-p-coumaroyl)-5-O-glucoside) and one Dp derivative (delphinidin 3-O-rutinoside (trans-p-coumaroyl)-5-O-glucoside) (in bold) are quantitatively abundant in black goji [22]. Consequently, Pt derivatives are the predominant BGAs and account for >95% of the total BGAs in fresh black goji [7,11,19]. Among all derivatives, Pt3R5G is the main component and accounts for almost 80% of the total BGAs [11,40]. The content of Pt3R5G in ripened black goji ranges from 8.29 to 31.51 mg/g DW, with an average value of 17.24 mg/g DW [1,36]. Due to the high concentration of Pt3R5G, the total BGA content of black goji is very high, and in fresh fruit, it reaches 24.04 mg/g fresh weight (FW), which is higher than other pigmented plants, such as blue honeysuckle (1.16–14.00 mg/g FW), blueberries (4.38–6.62 mg/g FW) and mulberry (0.12.73–3.83 mg/g FW) [15]. The dominance of Pt glycosides is also a unique feature of BGAs. In addition, there are many cis-trans isomeric anthocyanins in black goji. Acyl groups in acylated anthocyanins are typically trans configured, and only a few isomers are present in cis forms in nature [52]. The co-existence of both cis and trans anthocyanin isomers in the same source is another unique feature of BGAs.

2.3. Biosynthesis of BGAs

The biosynthesis of phenolic compounds in plants is controlled by genes of the corresponding enzymes [53]. The anthocyanin biosynthetic pathway in black goji is presented in Figure 1B, in which the transcription factors, including bHLH, MYB and WD40, forming a BMW tricomplex [54], co-regulate the transcription of structural genes such as flavonoid 3′hydroxylase (F3′H), flavonoid 3′5′hydroxylase (F3′5′H), anthocyanidin synthase (ANS), dihydroflavonol 4-reductase (DFR) and glucosyltransferase to control BGA biosynthesis [17,55,56,57]. The enhanced anthocyanin gene transcripts (F3′5′H, ANS, DFR and glucosyltransferase) and the increased ratio of F3′5′H/F3′H transcripts account for the biosynthesis and accumulation of Pt derivatives in black goji [10,56], where BGAsN1b and BGAsN2, encoding MYB transcription factors, could be responsible for the Pt3R5G biosynthesis by activating the pathway and regulating the accumulation, respectively [1,55,56,57].

While a plant’s anthocyanin profile is dependent on its genetic information, geographical conditions also play an important role in the anthocyanin structures [53]. The special geography (high altitude) and harsh weather conditions (low temperature, less rain and strong sunlight) of northwestern China, especially Qinghai-Tibet Plateau, dramatically promote the expression of glycosyltransferase HG27071 [22] and ultimately influence the BGA composition by linking sugar moieties and acyl groups to the anthocyanidins, forming glycosylated and acylated anthocyanins [2,18]. The glycosylation and acylation substitution in turn stabilize the chemical structures of BGAs during biosynthesis and accumulation [11], as well as during extraction, industrial processing and gastrointestinal digestion [6,22]. It is important for black goji to survive in such an area with special geography and harsh weather conditions.

Considering the discussion above, black goji is an ideal plant for agriculture and ecology in northwest China, encompassing Qinghai and Gansu Provinces and Inner Mongolia, Xinjiang Uygur and Ningxia Hui Autonomous Regions. The provinces or regions have significant differences in geographical conditions, including altitude, annual mean temperature and rainfall [2]. The anthocyanin composition patterns of black goji from different provinces or regions are the same, but the contents of Pt3R5G and total BGAs in black goji from different provinces or regions exhibit significant differences [2,25]. Meanwhile, black goji from the same province or region does not exhibit significant differences in the content of Pt3R5G and total BGAs [2]. This phenomenon evidences the important impact of geographical conditions on the content of BGAs. The correlation between the Pt3R5G contents and altitude is positive (r = 0.487, p < 0.01), while the correlations between the Pt3R5G contents and temperature and humidity are negative (r = −0.509, p < 0.01 and −0.377, p > 0.05 respectively) [1]. As a result, black goji from Qinghai, which has the highest mean altitude (2800 to 3000 m), lowest annual mean temperature (−5 to 5 °C) and annual mean precipitation (17 to 49 mm), possesses higher contents of both Pt3R5G and total BGAs than those from other provinces or regions in the order of Qinghai > Gansu > Inner Mongolia > Xinjiang > Ningxia [2]. Specifically, the content of total BGAs is increased with higher altitude, due to changes in temperature and humidity conditions. The content of Pt3R5G in black goji is also positively affected (r = 0.026, p > 0.05) by the annual mean sunshine hours, being beneficial to the seedling and growth of black goji [1,58]. The findings support the deduction of Wang et al. [59] that black goji from Qinghai has a higher market value compared to that from other provinces.

Due to the important impact of geographical origin on the content of BGAs, BGAs in turn could be used as an indicator for determining the authenticity of the geographical origins of black goji, which is important in quality control [59]. Wang et al. [2] adopted the data of individual BGA concentrations together with multivariate statistical analysis techniques to differentiate black goji from different geographical areas. The results of the principal component analysis (PCA) and linear discriminant analysis (LDA) provide a 100% successful differentiation rate. Liu et al. [36] observed a similar result, showing that PCA and the hierarchical cluster analysis (HCA) carried out using the content of individual BGAs present a clear separation of black goji according to geographical origins. In addition, by adopting the data of BGA concentrations obtained by near-infrared (NIR) spectroscopy and chemometrics, synergy interval partial least squares (Si-PLS), LDA, K-nearest neighbors (KNN), back propagation artificial neural network (BPANN) and least-squares support vector machine (LS-SVM) regression were systematically evaluated and compared during the development of a determinant model of black goji geographical origin and variety characterization [60]. The recognition rate of LS-SVM was >98.18%, showing excellent generalization for identification results.

On the other hand, the geographical conditions of Qinghai Province suitable for the biosynthesis and accumulation of these anthocyanins are limited, resulting in commercial devaluation and nutritional depreciation of black goji. Anthocyanins are specialized metabolites, the accumulation of which requires elicitors, and ethylene is an enhancer of anthocyanin contents during the growth of berries and vegetables. Foliar-applied ethephon (an ethylene-generating compound) on black carrot increased the anthocyanin contents by 25% [61,62]. Ethephon treatment by immersion of developing fruit of strawberry and ‘Rubi’ table grape also resulted in ripe fruit having greater anthocyanin contents [63,64]. The improvements could be that ethylene caused an increased expression of genes and enzymes as well as transcription factors related to the biosynthesis and accumulation of anthocyanins [61,64]. Ethylene also enhanced the acylation of anthocyanins (i.e., more stable), which protects plants from UV-B damage and subsequent cell death [11,62]. Moreover, postharvest ethylene treatment increased the anthocyanin contents in Cesanese red wine grapes by 17% [65]. Therefore, black goji from other provinces could be regulated by ethylene both in field and postharvest stages to obtain a higher content of BGAs.

2.4. Factors Influencing the Composition of BGAs

2.4.1. Degree of Ripeness

Anthocyanin biosynthesis can be influenced by various factors that result in the accumulation of anthocyanins in the specific cells or organs at particular developmental stages, leading to different anthocyanin levels in fruits at different growth stages [17,66]. The dynamic change in BGAs at three different growth stages (juvenile, expanding and mature) has been studied [1]. It was observed that the biosynthesis and accumulation of Pt3R5G are slow at the juvenile stage but increase significantly during the fruit expanding stage, especially at maturity. Compared with the juvenile stage, the Pt3R5G concentration is increased nearly 3- and 11-fold at the next two development stages, respectively. The increased level of anthocyanins is attributed to the high transcript levels of anthocyanin-related genes and regulators during the ripening process of black goji [67]. Among them, HG27071 and BGAsN2 gene expressions share a similar trend to Pt3R5G concentration during the development of black goji [1,22]. Specifically, a negative correlation (−0.072) is observed between BGAsN2 expression level and Pt3R5G concentration in the early green fruit stage [1]. This may be possible because the structural genes associated with the anthocyanin biosynthetic pathway (Figure 1B) might have not been effectively activated at the juvenile stage, leading to down-accumulation of anthocyanins [68]. However, the correlations are positive at the later stages (expanding, r = 0.344 and mature, r = 0.544) [1]. It is speculated that the synergistic effect of BGA biosynthesis, regeneration and translocation occurs primarily at the mature stage. These findings are helpful in determining the right time for black goji harvesting, which is important to ensure the quality of black goji.

2.4.2. Variety

In recent years, wild black goji resources have decreased dramatically due to destructive picking and the deterioration of the ecological environment [1]. Therefore, cultivated black goji were developed to meet the increased market demands [1,6]. Cultivated black goji exhibits the same profile of anthocyanins as wild black goji [2,6]. The reason is that cultivated black goji is basically transplanted from wild black goji, leading to an identical genetic profile for the two forms [6]. Interestingly, cultivated black goji has a relatively greater fruit size as compared to wild black goji [69]. Positive correlations are observed between anthocyanin contents and the fruit size of black goji [1]. Thus, it is speculated that cultivated black goji may have more BGAs than wild black goji. However, it was reported that the contents of both Pt3R5G and total BGAs are significantly lower in cultivated black goji than in wild fruits [1,6]. The reason is that cultivation has negative effects on the biosynthesis and accumulation of BGAs. The negative effects are caused by routine irrigation, fertilization, weeding and other cultivation operations [70]. The findings suggest that cultivar is important for black goji in biosynthesizing and accumulating more BGAs. Gamage et al. [24] proposed that greenhouse technology could be a suitable approach to produce new varieties of black goji which can be grown in a diverse range of climatic and soil conditions while preserving the high anthocyanin content in their fruit. Precise molecular breeding techniques, based on the characterization of the structural genes involved in BGA biosynthesis (Figure 1B), are also an appropriate approach to produce a type of black goji with a high anthocyanin yield.

2.4.3. Processing Techniques

Dried black goji is the most common product of black goji on the market, and oven-dried black goji has become more prevalent as thousands of drying units have been built in recent years by local governments [25]. The effects of different drying techniques on BGAs have been studied [2,25,71]. Three methods, i.e., freeze-drying (−40 °C), air-drying (room temperature, 22 to 25 °C) and oven-drying (50 °C), were compared. Freeze-dried black goji exhibits the same anthocyanin profile as the fresh black goji, but has less Pt3R5G (64.08%) than the fresh black goji (80%), which may occur during the transportation from the remote producing areas to the specialized laboratories, which takes about 8 h [21]. However, air- and oven-dried black goji show significant differences in both profiles and amounts of anthocyanins as compared to fresh and freeze-dried black goji. The content of Pt3R5G in BGAs from air- and oven-dried black goji was 11.79% and 0.72%, respectively, significantly lower than that of fresh and freeze-dried black goji [25,71]. Additionally, all anthocyanins in fresh and freeze-dried black goji have the structure of 5-O-glucoside, while most in air- and oven-dried black goji have no 5-O-glucoside structure [25,71]. Meanwhile, Cy and Pg derivatives were only found in oven-dried black goji [25], whereas the mechanism remains unclear. It is suggested that freeze-drying maximizes the retention of BGAs, and the thermal processing, i.e., air-drying and especially oven-drying, can cause severe structural changes and degradation of BGAs, as documented by Lu et al. [72] and Wang et al. [73]. This is understandable because anthocyanins have poor stability under light and high temperature conditions [74].

However, a contrary conclusion was reported, showing that temperature has a limited effect on BGA contents [75]. The mechanism was explored by studying the effects of acylation on the thermostability as well as the photostability and pH stability of BGAs [18]. Two individual BGAs, Pt3R5G and Pt 3,5-O-diglucosides (non-acylated), were compared, showing no significant differences for Pt3R5G under various temperature and light conditions, while the degradation of Pt 3,5-O-diglucosides is remarkable. In addition, only 15% of Pt 3,5-O-diglucosides and almost 50% of Pt3R5G are retained at pH 5 and 7, while more than 90% of the two anthocyanins are retained at pH 1 and 3. It is suggested that acylation significantly boosts the stability of BGAs not only in an acidic environment but also at neutral and alkaline pH, and lower pH (1 to 3) conditions are more favorable for the extraction or processing of BGAs, as documented by Tang and Giusti [33] and Shen et al. [41].

The effects of purification on the thermostability of BGAs have also been studied [37]. The thermostability of purified BGAs, unpurified BGAs and purified BGA–whey protein isolate (WPI) synthesis were compared, showing that they follow the order of purified BGA–WPI synthesis > purified BGAs > unpurified BGAs. It is suggested that the purification as well as combination with other substrates can improve the thermostability of BGAs, as highlighted by Qin et al. [76]. Inspired by this, chitosan (CS, 0.2–0.3 mg/mL) and casein phosphopeptide (CPP, 0.5%) composite gel system are used to prepare BGAs-loaded CS-CPP nanoparticles [77], and arabic gum (1%) and β-CD (50%) or gelatin (7%) and β-CD (40%) are used as the coating wall to microencapsulate BGAs [78,79]. The BGA nanoparticles and microencapsulates exhibit higher stability than the native BGAs when exposed to light, air and temperature. The co-pigmentation by glycosylation with glucose or polyphenol extracts is also likely to enhance the stability of BGAs [80]. It was reported that the addition of glucose in the freeze-dried black goji and the addition of polyphenol extracts from raspberry, Lonicera edulis and blackcurrant in the BGA solutions extend the half-lives of BGAs [73,81].

3. Color Properties of BGAs

The food industry has shown great interest in BGAs, owing to their attractive colors in food applications. BGAs exhibit a “red-purple-blue” pattern of color expression as pH increases from pH 2 (acidic) to pH 10–13 (alkaline) (Figure 2A,B) [31,82]. Red hue colors are found in acidic conditions, and vivid purple, blue and greenish-blue colors are found in neutral to alkaline environments. The pH-dependent transformation in the structure configuration explains the color patterns. As pH shifts from acidic to mild acidic or neutral conditions, the red flavylium cation either undergoes hydration and turns into colorless carbinol pseudobase, or bears deprotonation and exists in a blue-purple quinoidal base form. When the pH further increases, the quinoidal base could be ionized and becomes one or two negatively charged forms [83]. It is demonstrated that BGAs are promising natural colorants, producing various vivid hues over a wide range of pH.

Although the overall “red-purple-blue” pattern is the same, the color properties of BGAs are influenced by purification and acylation, in that there are pronounced discrepancies among the BGA extracts and isolates throughout the pH range tested (Figure 2C) [33]. Purification attenuates the saturation of color expression of BGAs, which is because the removal of interfering polyphenolics can destroy the inter-molecular co-pigmentation between these polyphenolics and BGAs. Acylation not only strengthens the color retention in acidic conditions, but also enhances the tinctorial strength and stability of BGAs in alkaline conditions, which is due to the forming of a folded structure called intra-molecular co-pigmentation between acyl groups and chromophore. Additionally, it was found that the color stabilities of BGA extracts and isolates are superior at pH 8 than in any other pH environment (Figure 2C) [33]. It was reported that dissociation constants for the structural transformation from quinoidal base to one negatively charged form and from one to two negatively charged forms of petunidin aglycones are pH 6.99 and 8.27, respectively [84]. Although these two numbers would not be the same as the dissociation constants of the isolated pigments Pt3R5G, it is still rational to postulate that a larger proportion of two negatively charged quinoidal base forms exists at pH 8, and therefore, Pt3R5G was found to be more resistant to degradation in these conditions. The analysis of colorimetric and spectrophotometric properties of BGAs also suggested that Pt3R5G contributes most of the color expression of BGAs, and shows superior color stability compared to other extracts over time [33]. This demonstrates the potential of Pt3R5G as a stable blue-hue natural colorant in food applications.

As limited natural pigments are available to match the color traits of synthetic ones, especially for blue and green hues [85], BGAs, especially Pt3R5G, would expand the choices of natural colorants in the food industry. However, little work has been reported on the application of BGAs as a natural food colorant in food systems [27]. Even so, BGAs expand the use of black goji, leading it to be used as a raw material to manufacture functional fermented beverages (called kombucha) [86], herbal tea [87] and natural vinegar [88]. The products are colorful and show a higher antioxidant capacity, total polyphenolic content and sensory acceptability than those made from red goji. It is noted that temperature, although it has limited effects on the content of BGAs, has significant effects on the color stability of BGAs during storage [75]. Specifically, a high storage temperature (37 °C) attenuates color intensity, and a low storage temperature (4 °C) causes more color changes. Therefore, it is preferable to use a low temperature in the manufacturing and especially storage of the foods.

Due to BGAs’ pH-dependent color expression, as described above, they could also be used to prepare pH-sensitive colorimetric packaging films in food applications for monitoring the freshness of foods. Starch–BGA films, prepared using cassava starch and BGAs (<4%) based on forming hydrogen bonds, show higher water vapor permeability, tensile strength, light barrier properties and antioxidant potential and are able to change colors in different buffer solutions (pH 2 to 13), thus exhibiting remarkable color variations with the change in pork quality (Figure 2D) [31]. The κ-carrageenan-BGAs films, prepared by incorporating κ-carrageenan with BGAs (<2.5%), show higher antioxidant activity, thermal stability and water vapor barrier properties and are also able to indicate the pH change in the media at a wide range of pH (2 to 10), thus exhibiting adequate capacity in monitoring the freshness of milk and shrimp (Figure 2E) [82].

4. Health Benefits of BGAs

4.1. Antioxidant Activities

BGAs are demonstrated to possess high antioxidant activity by several in vitro chemical assays, such as scavenging activities against 1,1-diphenyl-2-picrylhydra-zyl (DPPH), OH, O₂⁻ and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) free radicals, ferric-reducing antioxidant power (FRAP) and hydrophilic oxygen radical absorbance capacity (H-ORAC) [6,28,36,40,41,89,90]. The average values of DPPH, ABTS, FRAP and H-ORAC assays were about 41.87, 101.11, 90.62 and 4557 μmol Trolox equivalents/g [6,28]. The scavenging percentage of DPPH and ABTS were up to 85.97% and 69.81%, respectively, at a concentration of 100 μg/mL [40]. The high antioxidant activity of BGAs is mainly due to its unique anthocyanin profile with predominant 3,5-diglycosyl-anthocyanins. For flavonoids, the hydroxyl groups induce the antioxidant activities while the methylation of hydroxyl groups diminishes the antioxidant properties [91]. Pt has more hydroxyl groups than the anthocyanin aglycone of Pg, and the methylation of its hydroxyl groups is less than that of Mv (Figure 1A), leading to its high scavenging activity against radicals, such as superoxide and peroxynitrite, among the six most common anthocyanidins [46]. This is in line with the mechanism that anthocyanins act as potent antioxidants by donating hydrogen atoms to highly reactive free radicals and break the free radical chain reaction [11]. The acylation substitution also contributes to the antioxidant activities of BGAs, as acylated anthocyanins were observed to exhibit higher antioxidant activity than the nonacylated ones [18].

The reported antioxidant assays are usually conducted with crude BGA extracts. Extraction methods/solvents have a significant impact on the antioxidant capacity of the resulting BGA extracts. Wang et al. [40] observed that BGAs extracted by subcritical water show significantly higher antioxidant activities (DPPH and ABTS assays) than those extracted by conventional solvents (i.e., water and methyl alcohol). Moreover, the presence of other phenolic compounds or polysaccharides in the crude BGA extracts can influence their antioxidant activity. Zheng et al. [11] observed that BGA content shows negative correlations with ABTS (r = −0.503) and FRAP (r = −0.598) assays of the BGA extract. As a result, the antioxidant activity of crude BGA extracts is less than the purified BGAs. Shen et al. [41] reported that the IC₅₀ values of purified BGAs on DPPH, ABTS, H-ORAC and superoxide radicals are less than the crude BGA extracts. The presence of many electron withdrawing groups in the structure of purified BGAs, which weakens the dissociation energy of the O-H bond [32], can also contribute to higher antioxidant activity. Hu et al. [18] and Tang et al. [32] showed that isolated Pt3R5G exhibits a higher scavenging activity against DPPH, ABTS and superoxide radicals than the crude BGA extracts. Additionally, it was reported that the purified BGAs exhibit better scavenging activity by using ABTS assay than DPPH assay [32], while the inhibition ratio against DPPH is significantly higher than ABTS for the crude BGA extracts [40].

Most of the reported antioxidant activities are evaluated by in vitro chemical assays, whose bioactivities and mechanisms are likely to be different in live cells, which accounts for the uptake, metabolism and location of antioxidants within cells [34]. Even so, BGAs have a strong capacity to scavenge free radicals in a cellular environment, and, most importantly, exhibit no significant cytotoxicity on cells. Tang et al. [32] reported that Pt3R5G is effective in the inhibition of the reactive oxygen damage induced by H₂O₂ in neuronlike rat pheochromocytoma line cells, reflected in the recovery of plasma membrane integrity, the decreased production of malondialdehyde and the increased secretion of antioxidant enzymes, including catalase, superoxide dismutase and glutathione peroxidase. Song et al. [92] reported that BGA-rich extracts protect LLC-PK1 porcine renal tubules cells from 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative damage. Ran et al. [77] observed that the BGA-loaded CS-CPP nanoparticles (release percentage of 24.3–64.2% at pH 7) significantly elevate the survival rate of EAhy926 cells exposed to oxidized low-density lipoprotein (ox-LDL).

4.2. α-Glucosidase Inhibiting Activity

BGAs are observed to inhibit the activity of α-glucosidase from Saccharomyce cerevisiae and Caco-2 cells (IC₅₀ values of 1.32–1.57 and 25.3 μg/mL, respectively) and exhibit no substantial differences compared with the same dose of acarbose [6,21]. It is suggested that BGAs could be a promising inhibitor against α-glucosidase to prevent hydrolyzing terminal (1→4)-linked short chain reducing sugars (e.g., maltose and oligosaccharide), reducing blood glucose levels. α-glucosidase is located in the brush border of the small intestine, where BGAs produce an inhibitory effect against the enzyme. Although gastrointestinal digestion triggers changes in both BGA structure and content, it improves the α-glucosidase inhibitory activity of BGAs by lowering the IC₅₀ values from 1.32–1.57 μg/mL to 0.20–0.48 μg/mL [6]. This is consistent with an earlier study reporting that anthocyanins have higher α-glucosidase inhibitory activities after being digested [93]. However, BGAs before and after gastrointestinal digestion share a similar fluorescence spectra and quenching constants (K_FQ, the binding affinity of a quencher to an enzyme) [6], suggesting that the glycol-ligands at the third position in the C ring and the fifth position in the A ring may not influence the binding capacity of BGAs with α-glucosidase. The constant substitutional group in flavonoids are the hydroxyl group at the fourth position in the C ring and the seventh position in the A ring [94], and the inhibition capacity of α-glucosidase increases by binding more hydroxyl groups at the third, fourth or fifth positions in the B ring of anthocyanins [3]. Consequently, it is speculated that the mechanism of BGAs in inhibiting the activity of α-glucosidase is that BGAs bind to α-glucosidase with the hydroxyl ligands at the fourth position in the C ring, the seventh position in the A ring or the third, fourth and fifth positions in the B ring of the aglycone.

4.3. Alleviating Insulin Resistance

Insulin resistance (IR) can be induced by impairing insulin signal transduction and results in an overproduction of glucose that cannot be effectively utilized [95]. The phosphatidylinositol 3-kinase/serine–threonine-specific protein kinase B (PI3K/Akt) signaling pathway is an important pathway for regulating hepatic IR, and its activation can effectively alleviate IR [95]. It was reported that BGAs recover the reduced phosphorylation of the insulin receptor substrate IRS2 in IR Hep-G2 cells (induced by high glucose and palmitic acid) and high-fat diet (HFD)-fed mice, leading to the activation of PI3K and its downstream effector Akt and thus activating the PI3K/Akt signaling pathway [21,96]. The activated PI3K/Akt signaling pathway phosphorylates various downstream factors such as glycogen synthase kinase-3β (GSK3β) and forkhead box protein O1 (FOXO1), which promotes the synthesis of glycogen and inhibits gluconeogenesis, respectively [97]. The activated PI3K/Akt signaling pathway also mediates the mitochondrial dysfunction of hepatic cells by reducing the accumulation of mitochondrial reactive oxygen species (ROS) and improving intracellular adenosine triphosphate (ATP) production and mitochondrial membrane potential (MMP) level, leading to an improved mitochondrial function [98,99]. As a result, it was observed that BGAs (25 μg/mL) are helpful in glycometabolism, i.e., recovering the reduced phosphorylation of GSK3β and FOXO1, reducing the mitochondrial ROS and restoring the ATP production and the MMP level in IR Hep-G2 cells, and exhibit no substantial differences compared with the same dose of metformin [21]. It is suggested that BGAs could be used as a potential therapeutic alternative to reduce blood glucose levels, and the potential mechanism is illustrated in Figure 3.

Hyperglycemia is a common symptom of type 2 diabetes mellitus (T2DM), one of the most critical global public health issues [3]. Therefore, reducing blood glucose levels is an important strategy to prevent and control T2DM. Based on the above evidence, BGAs could be used as an alternative to synthesized drugs such as acarbose and metformin in reducing blood glucose levels to prevent and control T2DM. This is in line with the current non-pharmacological approach for preventing and controlling T2DM and its complications by consuming polyphenol-rich foods in our daily diet [100]. BGAs could also be used as a useful therapeutic substance to prevent and control diabetes-related diseases such as diabetic cardiomyopathy, a major mortality risk for diabetes patients. It was reported that BGAs remarkably attenuate cardiac dysfunction, myocardial fibrosis, oxidative stress status and cell apoptosis in the heart tissues of diabetic rats [101]. The inactivation of the protein kinase C and activation of Akt is induced by BGA treatment, providing a molecular mechanism for the cardioprotective effect of BGAs.

4.4. Anti-Inflammatory Effects

Host-related factors such as respiratory bursts of immune cells can produce free radicals which cause oxidative stress damage to the tissues, resulting in an overexpression of nuclear factor-kappa B (NF-κB) and thus pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-1β) and interferon γ (IFN-γ) [102]. With strong antioxidant activities (as described in Section 4.1) that confer protection against inflammation [103], BGAs exhibit potent anti-inflammatory effects to block pro-inflammatory cytokines, including TNF-α, IL-6, IL-1β and IFN-γ [15,30,96]. Chronic inflammation is a critical factor in the development and progression of chronic diseases [104,105,106]. Therefore, it is believed that BGAs could be used to prevent and treat chronic inflammatory diseases.

Specifically, BGAs exhibit potential ameliorative effects on atherosclerosis, a common pathological basis for the induction of cardiovascular and cerebrovascular diseases with surpassed mortality rates of cancer. It was reported that the expression levels of NF-κB and vascular cell adhesion molecule-1 (VCAM-1) in aorta of atherosclerosis rats are significantly down-regulated by BGAs, leading to decreased levels of serum TNF-α and IL-6 [15].

BGAs also exhibit potential protective effects on ulcerative colitis, one of the major forms of inflammatory bowel disease. It was reported that Pt3R5G and crude BGAs significantly inhibit lipopolysaccharides (LPS), Toll-like receptors 4 (TLR4) and nod-like receptor protein 3 (NLRP3), which can stimulate the production of pro-inflammatory cytokines including TNF-α, IL-6, IL-1β and IFN-γ, leading to decreased levels of these cytokines in colitis mice induced by dextran sodium sulfate (DSS) [30]. It was also reported that Pt3R5G and crude BGAs significantly increase the intestinal integrity and the expression of intestinal tight junction-associated proteins, including zonulae occludens-1, occludin and claudin-1 (associated with impairment of epithelial barrier function and induction of inflammation) in colitis mice [30]. Consequently, the treatments of Pt3R5G and crude BGAs increased the body weight, feed quantity, solid fecal weight and colon length, and reduced the disease activity index (DAI) of colitis mice. This is reasonable, as polyphenols can be alternate traditional medicines, e.g., aminosalicylates and immunomodulators to prevent or treat chronic inflammatory bowel diseases [107].

Memory disorders represent another type of chronic inflammatory disease that can be relieved by BGAs. It was reported that BGAs improve memory disfunction, neuroinflammation and neurodegeneration caused by _D-galactose [14]. The mechanism is BGAs activate the receptor for advanced glycation end products (RAGE)/NF-κB/C-jun N-terminal kinase (JNK) pathway, suppressing the microgliosis and astrocytosis, reducing the overexpression of IL-1β, TNF-α and cyclooxygenase-2 (COX-2), and lowering the caspase-3 levels and the B-cell lymphoma 2-associated X protein/B-cell lymphoma 2 (Bax/Bcl2) ratio (Figure 4). Additionally, it was reported that BGAs protect neurovascular unit (NVU) in rats impaired by middle cerebral artery occlusion/reperfusion (MCAO/R). The mechanism is BGAs activate the NF-κB/JNK pathways, reducing the expression of IL-1β, IL-6 and TNF-α, improving the blood–brain barrier disruption and inhibiting apoptosis (Figure 4) [20]. The effects also provide an opportunity for preventing and treating stroke induced by cerebral ischemia or reperfusion injury [20], improving the learning capacity and memory after surgery [108] and minimizing memory impairment of Alzheimer’s disease [109].

Furthermore, treatment with BGAs is also an important strategy to alleviate IR and then prevent and control T2DM. It was reported that BGAs improve IR in HFD-induced mice [108]. The mechanism is that BGAs inhibit hepatic inflammation by reducing activation of the TLR4/NF-κB/JNK pathway in the liver tissues and ameliorate oxidative stress by activating the nuclear factor erythroid-2-related factor 2/heme oxygenase-1/NADPH quinineoxidoreductase-1 (Nrf2/HO-1/NQO1) pathway. Obesity is a leading health concern in the world; it is closely related to inflammation and oxidative stress and is typically accompanied by IR and T2DM [110]. Therefore, it is speculated that the diabetes-related diseases that could be controlled by BGAs include not only diabetic cardiomyopathy (as described in Section 4.3), but also obesity. The used drugs against chronic inflammatory diseases exhibit adverse side effects [24]. In this context, functional foods and medicines with anti-inflammatory effects should be developed based on BGAs.

4.5. Promoting Lipid Metabolism

Elevated lipid levels in the serum are the main risk factor for the development of atherosclerosis that ultimately results in cardiovascular and cerebrovascular diseases [24]. Due to the side effects such as liver poisoning caused by consuming synthetic drugs, a recently encouraged strategy to regulate conditions of lipidemia and to lower serum lipid levels is consuming natural extracts. High-density lipoprotein cholesterol (HDL-C) in serum helps to transport serum lipids back to the liver for metabolism, which in turn reduces vascular lipids [111]. BGAs are reported to cause a significant increase in HDL-C concentrations in atherosclerosis rats induced by vitamin D₃ and HFD [15]. BGAs are also reported to regulate the key enzymes, cholesterol 7α-hydroxylase1 (CYP7A1) and sterol regulatory element binding protein-2 (SREBP-2), controlling the cholesterol metabolism in the liver by down-regulating the expression levels of SREBP-2 and up-regulating the expression levels of CYP7A1 in the atherosclerosis rats [15]. The phenomenon contributes to the synthesis of total bile acid (TBA), thereby promoting the excretion of cholesterol and lowering serum cholesterol levels. The findings suggest that BGAs promote lipid metabolism by increasing the serum HDL-C level and regulating the body’s lipid metabolism-related pathways. Due to these effects, BGAs are effective in lowering both the serum lipid and cholesterol levels to maintain a healthy lipid level in the body, therefore exhibiting a potential anti-atherosclerosis effect and thus reducing the risk of cardiovascular and cerebrovascular diseases [15]. This is also reasonable as anthocyanins can significantly reduce the aortic cholesterol in hyperlipidemic mice [112]. In this context, the consumption of BGAs in raw form or incorporating BGAs in a functional form may be a suitable choice for preventing and controlling atherosclerosis and also cardiovascular and cerebrovascular diseases.

4.6. Modulating Gut Microbiota

Anthocyanins exhibit positive modulation effects on the gut microbiota [113]. BGAs are reported to modulate gut microbiota by promoting the growth of gut-beneficial bacteria and retarding that of bad bacteria [16]. Specifically, they increase the relative abundance of Roseburia, Lachnospiraceae, Ruminococcaceae, Muribaculaceae, Akkermansia and Bacteroides and decrease the growth rate of Prevotellaceae, Helicobacter and Desulfovibrionaceae [15,114]. Among them, Roseburia, Lachnospiraceae, Ruminococcaceae, Muribaculaceae, Akkermansia and Bacteroides are common short-chain fatty acids (SCFAs) producing bacteria that can anaerobically ferment dietary fibers into SCFAs, including acetic, propionic and butyric acids [115]. The SCFAs are directly absorbed into the bloodstream, leading to various positive influences, e.g., activating G protein-coupled receptors (GPRs), inhibiting histone deacetylases (HDAC), increasing intestinal tight junction mRNA and protein expression levels, reducing intestinal permeability, protecting intestinal barrier integrity and mitigating intestinal inflammation. The positive influences make BGAs mitigate colonic barrier dysfunction and inflammation, providing insight into the mechanism by which BGAs protect against obesity. Prevotellaceae is a trimethylamine lyase producing bacteria and Helicobacter and Desulfovibrionaceae are endotoxin producing bacteria, and the decrease in their abundance reduces the predisposing factors, e.g., trimethylamine N-oxide and lipopolysaccharide, for the development of atherosclerosis [114,116]. It is suggested that BGAs improve atherosclerosis also by modulating gut microbiota, and the mechanism is proposed in Figure 5.

Additionally, both purified (Pt3R5G) and crude BGAs are observed to reverse the relative abundance of bad bacteria of Porphyromonadaceae, Helicobacter, Parasutterella, Parabacteroides, Oscillibacter and Lachnospiraceae in colitis mice induced by DSS [30]. The modulation facilitates the production of SCFAs which attenuate the inflammatory bowel diseases by blocking proinflammatory cytokines and increasing the tight junction protein [117,118]. It is suggested that BGAs also exhibit protective effects on colitis by modulating gut microbiota. Pt3R5G shows better protective effects on the gut microbiota than crude BGAs [30]. The difference could be because crude BGAs have only a down-regulation effect on the bacteria that promote colitis, while Pt3R5G not only shows a down-regulation effect, but also presents an up-regulation effect on the bacteria that prevent colitis.

However, alterations during gastrointestinal digestion can result in severe structural changes and degradation of anthocyanins [119]. An in vitro study revealed that gastrointestinal digestion causes severe structural damage to BGAs by losing its glycoside moieties in the gut environment, leading to a lower level of total BGAs after the digestion (by 15%) than before the digestion [6]. It is suggested that gastrointestinal digestion could decrease the total amount of bioavailable BGAs, as documented by Tagliazucchi et al. [120], therefore having a negative impact on BGAs’ modulation on gut microbiota. The loss of BGAs occurs in the stomach, where anthocyanins show a higher transport efficiency (in its intact form) with the action of bilirubin transferase, and also in the intestine environment by converting to chalcone and semi-chalcone [21,121]. Soluble dietary fiber (SDF) is resistant to digestion and absorption in the human small intestine, which can effectively entrap anthocyanins in the food matrix through non-covalent binding interactions, therefore protecting them from structural changes and degradation in the harsh upper gastrointestinal environment [122]. It is speculated that encapsulation of BGAs using SDF is a suitable choice for protecting BGAs against structural changes and degradation during gastrointestinal digestion, as suggested by Fang and Bhandari [123] and Tang et al. [124], which is helpful in promoting BGAs to play a role in regulating gut microbiota.

4.7. Other Health Benefits

The individual anthocyanin Pt3R5G is observed to stimulate prostate cancer DU-145 cells to produce a large amount of ROS in a dose-dependent manner and thus induce cell apoptosis by activating phosphatase and tensin homology deleted on chromosome 10 (PTEN) and inactivating Akt and thus activating the caspase-3 apoptotic pathway, i.e., the PTEN/PI3K/Akt/caspase-3 pathway [125]. This is reasonable, as ROS can activate or inhibit related signaling pathways leading to cell apoptosis [126,127]. As a result, it was observed that Pt3R5G inhibits cell proliferation and promotes cell cycle arrest at the S phase, as well as reduces mitochondrial membrane permeability [125].

BGAs are observed to increase serum glucose, liver/muscle glycogen and the activity of superoxide dismutase and reduce the vitality of lactate dehydrogenase, the content of malondialdehyde, the levels of lactic acid and serum urea nitrogen of fatigue mice, leading to an extended exercise time, enhanced exercise endurance, increased sugar reserves, fewer free radicals and improved metabolism, thereby exhibiting anti-fatigue activities [128]. BGAs are also observed to compete with L-tyrosine for tyrosinase and combine with the complex of L-dopa and enzyme to reduce the production of dopaquinone, resulting in an inhibitory effect on tyrosinase monophenolase and tyrosinase diphenolase, and thus can be used to treat hyperpigmentation disorders [41].

The health benefits of BGAs and their potential effects in preventing/treating chronic diseases are summarized in Figure 6, which provides a scientific basis for the development and utilization of BGAs. Future research is needed to focus on applying this basic knowledge to the development of functional foods and potential medicines based on BGAs.

5. Conclusions

A total of 39 anthocyanins in black goji (BGAs) can be detected and identified, among which eight Pt derivatives (>95%), one Mv derivative and one Dp derivative are quantitatively abundant in the fruit (Table 1). Pt3R5G is the most abundant anthocyanin and accounts for 80% of the total BGAs. The anthocyanins are characterized by 3,5-diglycosylation, organic acid acylation and the co-existence of cis and trans isomers, which is different from the anthocyanins in other berries and vegetables. Each type of anthocyanin is associated with different activities, and the acylation of anthocyanins can improve their stability and biological activity. Consequently, BGAs possess various health benefits such as antioxidant activity, inhibiting α-glucosidase activity, alleviating IR, anti-inflammatory activity, promoting lipid metabolism and modulating gut microbiota, and thus are helpful in preventing/treating some chronic diseases such as T2DM, diabetes-related diseases, memory disorders, stroke, colitis, atherosclerosis, cardiovascular and cerebrovascular diseases, cancer and hyperpigmentation disorders (Figure 6). The therapeutic mechanism of the health benefits of BGAs was systematically summarized in this work, which will promote the development of functional foods and potential medicines based on BGAs. In addition to these health benefits, BGAs also possess several other unique characteristics, such as an attractive color with a “red-purple-blue” pattern of color expression in a wide pH range, attracting great interest from the food industry to use BGAs as natural colorants and to prepare smart food packaging materials (Figure 2). This review promotes in-depth research of BGAs and their product development.