Phytochemicals Derived from Catharanthus roseus and Their Health Benefits

Hong Ngoc Thuy Pham 1,2,* , Quan Van Vuong 1, Michael C. Bowyer 1 and Christopher J. Scarlett 1,* 1 School of Environmental and Life Sciences, Faculty of Science, University of Newcastle, Ourimbah, NSW 2258, Australia; vanquan.vuong@newcastle.edu.au (Q.V.V.); michael.bowyer@newcastle.edu.au (M.C.B.) 2 Faculty of Food Technology, Nha Trang University, No. 2 Nguyen Dinh Chieu Street, Nha Trang City 650000, Vietnam * Correspondence: hongngocthuy.pham@uon.edu.au (H.N.T.P.); c.scarlett@newcastle.edu.au (C.J.S.)


Introduction
Natural resources including herbal plants, which contain a large variety of phytochemicals promising as traditional medicine to treat chronic and infectious diseases, have been considered as safe and effective alternatives with fewer side effects compared to synthetic agents [1]. Amongst the plethora of medicinal plants identified, Catharanthus roseus (L.) G. Don (C. roseus) has been widely used to treat various diseases in many countries. The hot water extract of the dried C. roseus leaf has been used for the treatment of diabetes in Jamaica, Kenya and the West Indies or the hot water extract of the dried plant has been taken orally as complementary and alternative therapies for various types of cancers, heart disease and leishmaniasis in Peru [2]. More scientific evidence has proved the potential health benefits of individual phytochemicals extracted from this plant. Of note, vinblastine and vincristine from C. roseus, and their synthetic analogues, have been used in combination with other cancer chemotherapeutic drugs for treating advanced testicular cancer, breast and lung cancers [3].
The drying and extraction process are crucial steps prior to isolation and purification of bioactive compounds from plant material. Drying aims to remove moisture content and reduce water activity in plant material, thus inactivating the enzymes responsible for degrading many bioactive compounds, in plant material, thus inactivating the enzymes responsible for degrading many bioactive compounds, decreasing the rate of microbial growth and reducing the costs of transportation and preservation. However, the drying treatment has been found to have a significant effect on the retention of bioactive compounds and antioxidant capacity of plant material [4][5][6][7]. Therefore, selecting a suitable drying method is very important to maintain the high yield of bioactive compounds and antioxidant power within plant material; however, the economic aspect and the accessibility of drying equipment should also be considered. Similarly, the extraction process is also a key step to obtain bioactive compounds from plant materials. Extraction techniques have been reported to significantly affect the extraction efficiency and stability of bioactive compounds [8][9][10][11]. Hence, this paper provides a comprehensive review of different techniques of drying, extracting and isolating bioactive compounds from the target plant material, C. roseus, and discusses its potential health benefits along with its traditional use.

Taxonomy and Traditional Use of C. roseus
C. roseus is native to Madagascar and known as the Madagascar periwinkle. It is now grown in many countries and is a common decorative, easy growing and spreading perennial herb. The C. roseus plant is 30-60 cm tall with young pubescent branches. Its leaves are oblong or oblanceolate, membranous, entire, obtuse or mucronate, and have short petioles ( Figure 1). Inflorescence occurs at the axillary with 1-4 flowered cymes, and flowers are from white to red depending on the cultivar. The calyx tube is short, but the corolla tube is long, sparsely pubescent above and the throat is hairy within or below the stamens. The ovary is pubescent, long and the stigma is pentagonal. Its fruits are 15-25 mm long and have two follicles [12]. This plant has been used as a traditional medicine in many countries ( Table 1). The dried leaf or entire plant is boiled with water and then the extract is taken orally to treat diabetes in Northeast India, the Cook Islands, Australia, England, Thailand, Natal, Mozambique, Philippines, Dominican Republic, Jamaica, Northern Europe and Vietnam [13][14][15][16][17]. The aqueous extract of the leaf or the whole plant is also used by Cook Island and Vietnamese people [13,17], and Kenyans as complementary and alternative therapies for various types of cancer including throat, stomach and oesophageal cancers [18]. The people in the Kancheepuram District of Tamil Nadu, India mix the powder of C. roseus whole plant with cow's milk, which is taken orally to treat diabetes [19]. C. roseus root is dried, ground and decocted for curing urogenital infections in the Venda region, South Africa [20], gonorrhoea in Limpopo Province, South Africa [21] and stomach ache in the Mutirikwi area of Zimbabwe [22]. This plant has been used as a traditional medicine in many countries ( Table 1). The dried leaf or entire plant is boiled with water and then the extract is taken orally to treat diabetes in Northeast India, the Cook Islands, Australia, England, Thailand, Natal, Mozambique, Philippines, Dominican Republic, Jamaica, Northern Europe and Vietnam [13][14][15][16][17]. The aqueous extract of the leaf or the whole plant is also used by Cook Island and Vietnamese people [13,17], and Kenyans as complementary and alternative therapies for various types of cancer including throat, stomach and oesophageal cancers [18]. The people in the Kancheepuram District of Tamil Nadu, India mix the powder of C. roseus whole plant with cow's milk, which is taken orally to treat diabetes [19]. C. roseus root is dried, ground and decocted for curing urogenital infections in the Venda region, South Africa [20], gonorrhoea in Limpopo Province, South Africa [21] and stomach ache in the Mutirikwi area of Zimbabwe [22].

Major Bioactive Compounds Derived from C. roseus
A range of alkaloids (nitrogen-containing organic compounds other than amino acids, peptides, purines and derivatives, amino sugars and antibiotics) [23] have been found in C. roseus (Table 2). Alkaloids are broadly classified as heterocyclic or non-heterocyclic, depending on the chemical structure. Heterocyclic alkaloids are those that contain the nitrogen atom in the ring system, the size of which leads to further subclassification as pyrrole, pyrrolizidine, pyridine, piperidine, quinoline, isoquinoline, norlupinane or indole alkaloids. Non-heterocyclic alkaloids, which are also sometimes called proto-alkaloids or biological amines, are less commonly found in nature. These molecules have a nitrogen atom, which is not a part of any ring system such as ephedrine, cathinone and colchicine [23].      Besides well-known alkaloids, new phenolic compounds were also found in the C. roseus leaf, stem, seed and petal (Table 3).  1330.4 ± 10.8 [32,33] This plant has been found to possess a number of important bioactive components that greatly contribute to the herbal medicine industry; however, their amounts present in the plant are often low. Of note, the biosynthesis of plant secondary metabolites is affected by the biotic and abiotic factors. Particularly, environmental stresses have been found to stimulate the production of secondary metabolites including alkaloids because of the plant protective function. Hence, more in vitro and in vivo studies focus on increasing the amounts of these compounds through changing environmental conditions such as light, salinity, soil types and nutrients, drought and metal stress. In particular, Misra and Gupta [34] found that salinity (100 mM NaCl) stimulated indole alkaloid accumulation in the C. roseus leaves and roots. The amount of ajmalicine in C. roseus increased with the treatment of cadmium (concentration ranging from 0.05 to 0.4 mM) from 24 to 48 h and excretion into the culture medium, especially for cells at the mid-exponential growth phase [35]. The study of Binder et al. [36] indicated that UV-B light stimulated alkaloid production in C. roseus hairy root due to protein kinase Besides well-known alkaloids, new phenolic compounds were also found in the C. roseus leaf, stem, seed and petal (Table 3). 1330.4 ± 10.8 [32,33] This plant has been found to possess a number of important bioactive components that greatly contribute to the herbal medicine industry; however, their amounts present in the plant are often low. Of note, the biosynthesis of plant secondary metabolites is affected by the biotic and abiotic factors. Particularly, environmental stresses have been found to stimulate the production of secondary metabolites including alkaloids because of the plant protective function. Hence, more in vitro and in vivo studies focus on increasing the amounts of these compounds through changing environmental conditions such as light, salinity, soil types and nutrients, drought and metal stress. In particular, Misra and Gupta [34] found that salinity (100 mM NaCl) stimulated indole alkaloid accumulation in the C. roseus leaves and roots. The amount of ajmalicine in C. roseus increased with the treatment of cadmium (concentration ranging from 0.05 to 0.4 mM) from 24 to 48 h and excretion into the culture medium, especially for cells at the mid-exponential growth phase [35]. The study of Binder et al. [36] indicated that UV-B light stimulated alkaloid production in C. roseus hairy root due to protein kinase stimulation. Additionally, biomass and alkaloid yield in cell suspension culture of C. roseus was found to increase along with the increased total nitrogen and phosphate at an adequate pH of test culture medium because nitrogen is an important component of alkaloids, while phosphorus affects the synthesis of alkaloids [37]. A similar phenomenon was also found in other medicinal plants. Oueslati et al. [38] found that polyphenol content of the Mentha pulegium leaf irrigated with a nutrient solution containing 100 mM NaCl for 14 days increased more than 3-fold higher than that of the control. The levels of terpenes increased in the green leaves of the thyme plants in response to drought stress [39].

Preparation of Dried C. roseus
In previous studies, various drying methods, such as freeze drying, air drying, low-temperature drying, infrared drying or drying under the shade have been applied to prepare dried C. roseus for further recovery of bioactive compounds ( Table 5). The results showed that drying methods significantly affected the retention of bioactive compounds in C. roseus material because bioactive compounds are sensitive to heat, light and oxygen [40]. Freeze drying has been reported as a prominent and effective drying method in terms of the retention of bioactive compounds, but it is costly and not typically available in some of the regions where the plant is collected and processed [5]. Among thermal drying methods, infrared drying at 35 • C was found to be suitable for saponin retention within the C. roseus stem and root, while vacuum drying at 50 • C was suggested for drying the leaf and the flower, which contained high levels of phenolics and flavonoids [40]. However, there is no report on the effect of drying conditions on the individual bioactive compounds present in this plant material, suggesting future studies on investigating the optimum drying conditions for recovering target compounds from C. roseus are required.

Recovery of Bioactive Compounds from C. roseus
There have been numerous extraction techniques used for the recovery of bioactive compounds from C. roseus, including conventional extraction, ultrasonic-assisted extraction and Soxhlet extraction (Table 5) using the common solvents methanol and ethanol. Soaking materials with 90% ethanol and leaving the mixture overnight has been used to extract vindoline, catharanthine, vincristine and vinblastine from C. roseus leaf (Table 5) [42]. To obtain the phenolic extract, lyophilized and powdered plant material (leaves, stems, seeds and petals) were extracted with boiling water for 20 min [33]. In this method, the solvent diffuses into the material matrix, dissolves solutes and liberates them from the plant matrix. This is driven by a concentration gradient at the solid-liquid interphase that exists until the equilibrium is established [44]. This method is cheap and simple but requires prolonged extraction times that may lead to the loss of active compounds through oxidation, ionization and hydrolysis. The Soxhlet extraction was used to prepare the C. roseus hairy root extract for isolation of vindoline, ajmalicine, serpentine and catharanthine [41]. Although this method is simple, well established and inexpensive, it is also time consuming and requires large quantities of solvent. Further, it occurs at the boiling point of solvents over a long period that may cause the thermal degradation of bioactive compounds [45].
Ultrasonic extraction is an emerging extraction technique with numerous advantages over conventional thermal extraction methods including shorter extraction times, higher extraction yields, energy and solvent cost savings. C. roseus has been subjected to ultrasonic extraction to obtain specific target compounds (Table 5) [24,26,32,40,46]. Other advanced extraction techniques such as microwave-assisted extraction (MAE), enzyme-assisted extraction, pulsed electric field extraction, pressurised liquid extraction and supercritical fluid extraction, which can also reduce extraction time, solvent consumption and environmental pollution could be considered; however, industrial up-scaling potential needs to be taken into consideration.
To obtain phenolic extracts from C. roseus, Ferreres et al. [32] and Pereira et al. [33] applied different extraction methods that are ultrasound-assisted extraction and maceration using methanol:water (1/1) as the extraction solvent at room temperature for 17 h versus extraction with boiling water for 20 min (Table 5). In the latter method, water-a cheap, accessible and environmentally friendly solvent-was used, and shorter extraction time was applied as the extraction process was conducted at the boiling point, while the 50% aqueous methanol was used in the former one. However, the phenolic extraction efficiency of these methods could not be fully clarified as the quantity of individual phenolic compounds has not been determined in the former study. C. roseus crude extracts could be further separated using chloroform or using ion exchange resins to collect fractions that would be subjected to silica gel column chromatography to collect subfractions, then the individual compounds further purified using column chromatography over Sephadex LH-20 and preparative TLC or preparative HPLC [28,30,31,42]. The detailed recovery process of bioactive compounds from C. roseus is presented in Table 5. Of note, besides extraction techniques various extraction parameters, such as temperature, time, solvents, solvent-to-sample ratio, particle size, solvent pH and pressure have been found to significantly affect extraction efficiency and stability of bioactive compounds from plant materials [40,[46][47][48][49][50], future studies are recommended to investigate the optimal extraction conditions for recovery of groups or individual alkaloids and/or phenolic compounds from C. roseus for further extraction and isolation. In addition, advanced techniques should be applied to identify and test the potential use of unknown compounds in C. roseus.

Potential Use as an Anticancer Agent
C. roseus has been found to contain a range of alkaloids possessing anticancer activity including vinblastine, vincristine, vindoline, vindolidine, vindolicine, vindolinine and vindogentianine [27,43,51]. They inhibit cell proliferation through changing the microtubular dynamics, which induces apoptosis [52]. Of these, vinblastine and vincristine were the first plant-derived anticancer agents deployed for clinical use [51]. Vinblastine sulphate has been utilised for treatment of Hodgkin's disease, lymphosarcoma, choriocarcinoma, neuroblastoma, carcinoma of breast and lungs and other organs in acute and chronic leukaemia. Vincristine sulphate, an oxidised form of vinblastine [52] arrests mitosis in the metaphase and is effective in treating acute leukaemia (in children), lymphocytic leukaemia, Hodgkin's disease, Wilkins's tumour, neuroblastoma and reticulum cell sarcoma [2].
A range of other indole alkaloids extracted from C. roseus has been found to exhibit potent cytotoxic activity against various cancer cell lines. Catharoseumine, a new monoterpenoid indole alkaloid isolated from the C. roseus whole plant was found to possess an inhibitory effect on the human promyelocytic leukaemia HL-60 cell line with an IC 50 of 6.28 µM [28]. Moreover, three newly isolated dimeric indole alkaloids including 14 ,15 -didehydrocyclovinblastine, 17-deacetoxycyclovinblastine, 17-deacetoxyvinamidine and five known compounds (vinamidine, leurosine, catharine, cycloleurosine and leurosidine) possessed in vitro inhibition of cell viability against the human breast cancer cell line MDA-MB-231 with an IC 50 range of 0.73-10.67 µM [30]. Importantly, cathachunine, a new bisindole alkaloid from C. roseus, displayed antitumour effects on human leukaemia cells, but at much lower cytotoxicity against normal human endothelial cells, indicating that its activity inhibited selectively toward leukaemia cells.
Along with the studies on anticancer activity of individual alkaloids from C. roseus, effect of the entire crude extract on various cancer cell lines was also investigated. Recent findings found that C. roseus root and stem extract possessed strong in vitro cytotoxic activity against a panel of cancer cell lines [53,54]. Similarly, the study of Fernández-Pérez et al. [55] confirmed that the powerful antitumor activity of the indole alkaloid-enriched extract obtained from C. roseus cell cultures did not result from the effect of a single compound, but depended on the joint action of bioactive compounds. These results revealed the synergistic effect and positive interaction of the bioactive components present in C. roseus against cancer cells, which has also been found in other plant materials [56,57] and considered as a strategy for cancer treatment [58]. This phenomenon is possible due to the assistance of compounds with little or no activity to the main active compounds through helping the active component reach the target by improving bioavailability, decreasing metabolism and excretion, complementing mechanisms of action, reversal of resistance and modulating side effects [59].

Potential Use as an Antidiabetic Agent
C. roseus has been traditionally used as a treatment for diabetes in many regions of the world (Table 1). Juice from the leaf of C. roseus was reported to produce a dose-dependent reduction in blood glucose of both normal and diabetic rabbits [60]. The whole plant C. roseus methanolic extract displayed effective antihyperglycaemic activity, correlating with improvement in body weight, lipid profile and regeneration of β-cells of the pancreas in diabetic rats [61]. Recently, Tiong et al. [27] investigated the in vitro antidiabetic properties of four pure alkaloids including vindoline, vindolidine, vindolicine and vindolinine isolated from C. roseus leaf via the assays of 2-NBDG glucose uptake and inhibition of PTP-1B, an enzyme that regulates negatively the insulin signalling pathway. Improving glucose uptake in pancreatic or muscle cells could amend the hyperglycaemic conditions of type 2 diabetes. It was found that the four alkaloids increased the glucose uptake in mouse β-TC6 pancreatic and mouse myoblast C2C12 cells, coupled with their inhibition of PTP-1B. Among them, vindolicine showed the highest activity. The results supported the traditional utilisation of C. roseus for diabetic treatment, highlighting that C. roseus is a potent source for further exploring antidiabetic agents.

Potential Use as Anti-Alzheimer's Disease Agents
Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system and accounts for 50-60% of dementia in patients [62]. It is characterised by profound memory impairment, emotional disturbance and personality changes in the late stages of life [63]. Cholinergic neurons in the neocortex and hippocampus are suggested to be predominantly affected in AD, resulting in the cholinergic hypothesis, which associates AD symptoms to cholinergic deficiency. Therefore, symptomatic treatment for AD has been focused upon augmenting brain cholinergic neurotransmission [64]. The current effective AD therapy is to increase acetylcholine (Ach) levels by inhibiting acetylcholinesterase (AchE), an enzyme responsible for degrading Ach in the synaptic cleft [62,64]. The aqueous extract of C. roseus leaf, stem and root has been shown to effectively inhibit AchE in an in vitro microassay [33,65]. Additionally, serpentine, an alkaloid present in C. roseus leaf, stem and root [24] displayed a strong activity against AchE with a low IC 50 value (0.775 µM) [66]. These findings revealed that this plant is a potential source of active compounds for the pharmacological management of neurodegenerative conditions including Alzheimer's disease.

Other Beneficial Effects
Many microorganisms cause disease in plants, animals and humans, leading to the loss of crops, spoilage of food and increasing problems for human health [67]. Further, microbial resistance, which has become a rising challenging for agriculture and human health, requires more effective antimicrobial agents [52]. Ethanol extracts of C. roseus leaf, stem, flower and root have been reported to display antibacterial activity against various bacteria [68,69]. Additionally, the saponin-enriched fractions from C. roseus stem and root revealed their potent antifungal activity against Candida albicans and Aspergillus niger [53,54]. Moreover, individual compounds isolated from C. roseus were identified as potent antimicrobial agents. Yohimbine, an alkaloid isolated from C. roseus leaf, stem and root [24] was found to exhibit not only antibacterial, antifungal activities but also antiviral activity against herpes simplex virus (type 1) [70]. Remarkably, catharoseumine present in C. roseus whole plant was found to inhibit Plasmodium falciparum falcipain-2, a protozoan parasite that causes malaria, with an IC 50 value of 4.06 µM [28].
Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers in damaged tissue as closely as possible to its normal state. The ethanol extracts of C. roseus flower and leaf has been demonstrated to possess wound healing activity in rats [71,72], resulting from the astringent and antimicrobial properties of bioactive components present in the C. roseus extract [73]. In particular, the leaf extract promoted wound healing in diabetic rats [71]. The results coincided with the findings of Singh et al. [73] who found that the methanol extract of C. roseus leaf significantly increased wound contraction in streptozotocin induced diabetic mice. The wound healing activity of C. roseus extracts is promising to overcome the poor wound healing typical in the diabetic condition. In these instances, wounds commonly slow healing and can exist for weeks, raising significant challenges for wound care and control in clinical practice.
The leaf extract of C. roseus was shown to possess hypotensive and lipid lowering effects in adrenaline-induced hypertensive rats [74]. Ajmalicine found naturally in C. roseus [24] has been considered as a hypotensive agent as it acted as an α-adrenergic receptor antagonist [52,75]. Additionally, C. roseus extracts and isolated alkaloids including vindoline, vindolidine, vindolicine and vindolinine were found to possess antioxidant properties [27,46,53,54], which could reduce and prevent the oxidation of other molecules. Although oxidation reactions are crucial for life, they produce free radicals and then start a cascade of biochemical reactions, causing oxidative stress that damage or kill cells and contribute to cell ageing, cardiovascular disease, brain disease (associated with Alzheimer s disease), mutagenic changes and cancerous tumour growth [76,77].

Conclusions
C. roseus can be considered as a rich source of alkaloids and phenolics, which possess diverse biological properties including anticancer, antidiabetic, antioxidant, antimicrobial and antihypertensive activities. Numerous alkaloids and phenolics have been identified in this material but many compounds are unknown. Consequently, the identification and isolation of new phytochemicals within the different structural components of C. roseus should be continued. In addition, potential uses of bioactive compounds derived from this material need to be further investigated for applications in the nutraceutical and pharmaceutical industries.