Antioxidant, Anti-inflammatory Activities and Polyphenol Profile of Rhamnus prinoides

Rhamnus prinoides L’Herit (R. prinoides) has long been widely consumed as folk medicine in Kenya and other Africa countries. Previous studies indicated that polyphenols were abundant in genus Rhamnus and exhibited outstanding antioxidant and anti-inflammatory activities. However, there are very few studies on such pharmacological activities and the polyphenol profile of this plant up to now. In the present study, the antioxidant activities of the crude R. prinoides extracts (CRE) and the semi-purified R. prinoides extracts (SPRE) of polyphenol enriched fractions were evaluated to show the strong radical scavenging effects against 1,1-diphenyl-2- picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) (0.510 ± 0.046 and 0.204 ± 0.005, mg/mL), and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (0.596 ± 0.005 and 0.096 ± 0.004, mg/mL), respectively. Later, the SPRE with higher contents of polyphenols and flavonoids displayed obvious anti-inflammatory activities through reducing the NO production at the dosage of 11.11 − 100 μg/mL, and the COX-2 inhibitory activity with an IC50 value at 20.61 ± 0.13 μg/mL. Meanwhile, the HPLC-UV/ESI-MS/MS analysis of polyphenol profile of R. prinoides revealed that flavonoids and their glycosides were the major ingredients, and potentially responsible for its strong antioxidant and anti-inflammatory activities. For the first time, the present study comprehensively demonstrated the chemical profile of R. prinoides, as well as noteworthy antioxidant and anti-inflammatory activities, which confirmed that R. prinoides is a good natural source of polyphenols and flavonoids, and provided valuable information on this medicinal plant as folk medicine and with good potential for future healthcare practice.


Introduction
Rhamnus prinoides L'Herit (R. prinoides), belonging to family Rhamnaceae, and also commonly known as dogwood or Gesho in Amharic, is widely distributed in many countries of eastern, central, and southern Africa [1]. This plant has numerous valuable uses in local communities: the fruits are edible as a food source; the wood is hard and used as timber; and the stems and branches are usually made into ornaments, shades, fence edges, etc. [2]. Nowadays, as an important hopping agent in equivalents (GAE)/g and 352.25 ± 10.95 RE/g, which were eight times higher than that of dry R. prinoides powders at 26.86 ± 1.57 GAE/g and 41.46 ± 4.80 RE/g, respectively. Polyamide is a kind of polymer containing amide bonds (-CONH-), which can tightly absorb and bind with the compounds with hydroxyl phenols, acids, quinones, and nitro groups by the hydrogen bonds. Therefore, polyamide resin is particularly suitable for the purification and separation of the phenolic components from the complex natural products, including flavonoids, phenolic acids, quinones, carbonyl, and carboxyl compounds [24]. After purification with the polyamide resin in the present study, the TPC and TFC in semi-purified R. prinoides extracts (SPRE) went up to 553.67 ± 7.06 GAE/g and 958.21 ± 21.18 RE/g, showing an almost three times higher than that of in CRE. Based on the yields of CRE (11.72%) and SPRE (1.36%) to the R. prinoides powders, the higher TPC and TFC values in SPRE in this study also demonstrated the effective application of polyamide resin for the enrichment of the polyphenols. In this regard, the CRE (352.25 ± 10.95 mg RE/g extract) and SPRE (958.21 ± 21.18 mg RE/g extract) in this study obtained from R. prinoides in Kenya communities presented higher TFC values than that of the methanol extract (51.17 mg quercetin equivalent (QE)/g extract), aqueous extract (24.09 mg QE/g extract) and traditional boiling aqueous extract (12.03 mg QE/g extract) of Rhamnus alaternus bark from Algeria [25], and ethyl acetate (EA) extract (108.03 ± 3.09 mg catechin equivalent (CE)/g extract) of Rhamnus lycioides leaves from Algeria [26]. The distinct diversities of TFC values in the aforementioned extracts might be caused by the differences of extraction solvent polarities and purification processes.

Evaluation of Antioxidant Activities
The antioxidant activities of the CRE and SPRE were evaluated by the most frequently-used DPPH and ABTS radical scavenging tests, and all the results were expressed as IC 50 and Trolox equivalents (TEs) and shown in Table 2. For the DPPH assays, the SPRE at IC 50 of 0.204 ± 0.005 mg/mL exerted significantly higher scavenging activity than CRE of 0.510 ± 0.046 mg/mL (p < 0.01), together with the similar trend at the 2361.3 ± 57.9 µM TE/g and 945.5 ± 85.2 µM TE/g (p < 0.01). As for the ABTS assays, the SPRE also exhibited higher ABTS free scavenging activities compared with the CRE in regard to the IC 50 values (0.096 ± 0.004 vs. 0.596 ± 0.005, mg/mL) and TEs (1697.9 ± 70.7 vs. 273.5 ± 2.29, µM TE/g) (p < 0.01). Interestingly, the SPRE displayed higher DPPH scavenging activity of IC 50 at 0.204 ± 0.005 mg/mL and TE at 2361.3 ± 57.9 µM TE/g than the positive control butylated hydroxytoluene (BHT) of IC 50 at 0.286 ± 0.010 mg/mL and TE at 1684.3 ± 46.3 µM TE/g, which simultaneously confirmed the antioxidant potential of R. prinoides.
Similar studies revealed that the bark extracts of four Rhamnus species from Croatia communities, namely Rhamnus alaternus, Rhamnus fallax, Rhamnus intermedia, and Rhamnus pumila, displayed relatively lower DPPH values of 78.7 ± 3.16, 22.3 ± 0.54, 72.2 ± 4.00, and 53.1 ± 1.57 µg/mL [27], respectively, and also the bark extracts of Rhamnus catharticus and Rhamnus orbiculatus from Dedin and Mt. Sniježnica with the efficient concentration (EC 50 ) values of 64 ± 5 and 89 ± 2 µg/mL [28], respectively. Hence, the obtained antioxidant activities for the above extracts might be a result of the high TPC and TFC levels quantified in the extracts. As a matter of fact, phenolic compounds are commonly closely correlated with antioxidant activities, which might be due to their hydrogen-donating properties as free radical scavengers. Particularly, lots of well-known flavonoids, such as quercetin, kaempferol, isorhamnetin, rhamnetin, and their glycosides identified in the present study, might be attributed to those results [26]. As the active part, the H atoms in the hydroxyl groups of polyphenols can combine with the free radicals to form the polyphenol-radicals, and then react with other radicals, thus terminating the radical chain reaction [29]. In this current assay, it was assumed that the DPPH·and ABTS + free radicals in the solution turned into the non-radicals of the DPPH-H and ABTS, once encountering the H atom donors of hydroxyl groups of polyphenols in R. prinoides. That is to say, the active H atom donors, polyphenols, in R. prinoides possess strong antioxidant activity by capturing the DPPH and ABTS + free radicals. To this end, the SPRE was thus applied logically into the following assays.

Evaluation of Anti-inflammatory Activities
NO is an important inflammatory mediator secreted by activated macrophages. When produced in large quantities, NO will combine with O 2 to form peroxynitrite anion, which is an important factor leading to cell damage, energy depletion, and cell death, as well as an important link for NO to produce pathological damages [30]. COX-2 is known as a key rate-limiting enzyme that catalyzes the biosynthesis of the prostaglandins (PGs) from the precursor compound of the arachidonic acid, which induces the inflammatory cells to release chemokines and promote the movement of the inflammatory cells, thus facilitating the occurrence and metastasis of the inflammation [31]. Considering that inhibition of COX-2 activity and NO production could reduce damages to normal cells in the process of inflammation, thus significantly alleviating the typical inflammatory symptoms, they have become two of the major targets for the treatment of inflammatory diseases. Therefore, drugs that could inhibit COX-2 activity and NO production have potential anti-inflammatory activity. According to Figure 1a, compared with the negative group (NC) group, the NO production was significantly decelerated when the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells were incubated with the SPRE of R. prinoides at the concentrations of 11.11 -100 µg/mL in a dose-dependent manner. Meanwhile, the SPRE in Figure 1b also exhibited an obvious inhibitory effect against COX-2 with an IC 50 value of 20.61 ± 0.13 µg/mL, compared with the positive control aspirin at an IC 50 value of 6.33 ± 0.05 µg/mL. In previous studies, the R. davurica leaf extract demonstrated remarkable anti-allergic activity by getting involved in the Fyn/Syk pathway in the antigen-stimulated mast cells [32]; quercetin from Rhamnus nakaharai and frangulin B from Rhamnus formosana displayed strong inhibition against the formation of TNF-α in the LPS-stimulated RAW 264.7 macrophage cells with IC 50 values of 49.7 ± 6.1 µM and 24.2 ± 12.8 µM, respectively. Meanwhile, flavonoids of quercetin, quercetin 3-O-methyl ether, kaempferol from R. nakaharai, and frangulin B from R. formosana also displayed strong inhibition against the formation of TNF-α in the LPS/IFNγ-stimulated N9 cells with IC 50 values of 85.6 ± 2.2 µM, 43.3 ± 10.1 µM, 11.0 ± 4.6 µM, and 42.6 ± 2.8 µM, respectively, compared with the positive control of dexamethasone at an IC 50 value of 82.0 ± 3.8 µM [33]. In addition, 11 compounds were further screened out to be the potent COX-2 inhibitors from the bark extract of R. davurica with the UF-HPLC-MS hyphenated technique, in which vitexin, apigenin, and kaempferol exerted outstanding COX-2 inhibitory effect with IC 50 values of 55.94 ± 2.59, 10.14 ± 0.45 and 9.27 ± 0.43 µg/mL, respectively [34]. However, there is no such report about the anti-inflammatory applications in floks of R. prinoides in Kenya until now. Evidences from the HPLC-MS analysis in Figure 2 and Table 3 revealed that the above-mentioned flavonoids and their glycosides were also contained in the R. prinoides extracts from Kenya communities. For the first time, hence, the present results proved the noteworthy anti-inflammatory effects of R. prinoides by inhibiting the COX-2 activity and NO production in RAW264.7 cells. According to Figure 1a, compared with the negative group (NC) group, the NO production was significantly decelerated when the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells were incubated with the SPRE of R. prinoides at the concentrations of 11.11 -100 μg/mL in a dose-dependent manner. Meanwhile, the SPRE in Figure 1b also exhibited an obvious inhibitory effect against COX-2 with an IC50 value of 20.61 ± 0.13 μg/mL, compared with the positive control aspirin at an IC50 value of 6.33 ± 0.05 μg/mL. In previous studies, the R. davurica leaf extract demonstrated remarkable anti-allergic activity by getting involved in the Fyn/Syk pathway in the antigen-stimulated mast cells [32]; quercetin from Rhamnus nakaharai and frangulin B from Rhamnus formosana displayed strong inhibition against the formation of TNF-α in the LPSstimulated RAW 264.7 macrophage cells with IC50 values of 49.7 ± 6.1 μM and 24.2 ± 12.8 μM, respectively. Meanwhile, flavonoids of quercetin, quercetin 3-O-methyl ether, kaempferol from R. nakaharai, and frangulin B from R. formosana also displayed strong inhibition against the formation of TNF-α in the LPS/IFNγ-stimulated N9 cells with IC50 values of 85.6 ± 2.2 μM, 43.3 ± 10.1 μM, 11.0 ± 4.6 μM, and 42.6 ± 2.8 μM, respectively, compared with the positive control of dexamethasone at an IC50 value of 82.0 ± 3.8 μM [33]. In addition, 11 compounds were further screened out to be the potent COX-2 inhibitors from the bark extract of R. davurica with the UF-HPLC-MS hyphenated technique, in which vitexin, apigenin, and kaempferol exerted outstanding COX-2 inhibitory effect with IC50 values of 55.94 ± 2.59, 10.14 ± 0.4,5 and 9.27 ± 0.43 μg/mL, respectively [34]. However, there is no such report about the anti-inflammatory applications in floks of R. prinoides in Kenya until now. Evidences from the HPLC-MS analysis in Figure 2 and Table 3 revealed that the above-mentioned flavonoids and their glycosides were also contained in the R. prinoides extracts from Kenya communities. For the first time, hence, the present results proved the noteworthy anti-inflammatory effects of R. prinoides by inhibiting the COX-2 activity and NO production in RAW264.7 cells.

HPLC-UV/ESI-MS/MS Analysis
A chemical fingerprint profile can comprehensively reflect the types and quantities of chemical components contained in medicinal plants and their products, and then describe and evaluate their quality as a whole. Therefore, it can be used to analyze the authenticity, goodness, and stability of the quality of medicinal plants and their products [23]. By combining the high separation performance of HPLC to complex samples, with high selectivity, sensitivity, and the ability to provide molecular weight and structure information of MS, the hyphenated HPLC-MS is very   Table 3 below.

HPLC-UV/ESI-MS/MS Analysis
A chemical fingerprint profile can comprehensively reflect the types and quantities of chemical components contained in medicinal plants and their products, and then describe and evaluate their quality as a whole. Therefore, it can be used to analyze the authenticity, goodness, and stability of the quality of medicinal plants and their products [23]. By combining the high separation performance of HPLC to complex samples, with high selectivity, sensitivity, and the ability to provide molecular weight and structure information of MS, the hyphenated HPLC-MS is very suitable for the comprehensive evaluation of the QC of medicinal plants [35,36]. R. prinoides has been widely used in Kenyan local communities to treat a variety of diseases. However, studies on the chemical compositions of this plant and its products remain rare, and the lack of research in this field cannot guarantee the clinical efficacy of this plant and its products. Therefore, current studies are urgently needed to analyze the chemical constituents of this medicinal plant comprehensively.
In present study, the HPLC-UV chromatogram profile of R. prinoides (Kenya) was implemented by using the HPLC-UV/ESI-MS/MS. As shown in Figure 2 above, 65 peaks were detected in SPRE. Further structural identification and characterization of those compounds in Table 3 were carried out strictly by comparison with the chemical standards or the MS fragments reported in previous studies [12][13][14][15]23,37,38]. As a result, several types of polyphenols, including flavonoids and their glycosides (peaks 3-9, 11-15, 18, 19, 21, 22, 24-26, 28-31, 34, 37, 38, 40-42, 44-50, 53, 54, 56-63, 65), phenols and their glycosides (peaks 1, 2, 20, 23), naphthols and their glycosides (peaks 16,36,39,43,51,52), anthraquinones and their glycosides (peaks 17, 33, 35, 64), phenylpropanoid glycoside (peak 55), and saponin (peak 32), are revealed majorly in R. prinoides. On this basis, further studies on spectrum-effect should also be carried out to truly correlate its QC with its clinical efficacy, and help to clarify the mechanisms of action. For the relative quantitative analysis of chemicals in SPRE, the aforementioned flavonoids, phenols, naphthols, anthraquinones, phenylpropanoid, saponin, and their glycosides take up 69.54%, 2.39%, 17.87%, 6.39%, 0.53%, 0.98%, respectively. Most interesting, geshoidin, a naphthalenic glucoside, holds the highest content, which might be the reason that it acts as the basic bittering ingredient for the popular beverages of Tella and Tedj in Ethiopia and Cameroon [3]. Besides, geshoidin also exhibited outstanding superoxide anion (O 2 •-) scavenging activity in vitro with an IC 50 value at 1.9 mM, compared with the ascorbic acid (Vc) at 1.7 mM [16]. Therefore, for the polyphenol profile of R. prinoides, in this regard, both the flavonoids profiling of the main components and the highest ingredient of geshoidin (iconic marker) strongly provide the scientific basis for the quality assurance and functional components of this medicinal and edible plant. Coincidentally, our earlier study also indicated that antioxidant activities correlated closely with the higher content of flavonoids in R. davurica [23]. In other words, the higher TFC values in SPRE further demonstrated the potent stronger antioxidant and anti-inflammatory activities of R. prinoides in the present study.

Plant Material and Sample Preparation
The fresh stems and stem barks of R. prinoides (3.0 kg) were collected randomly from a 10,000 m 2 area of Mount Kenya (Kenya) in July of 2018. Afterward, the specimens of those plant materials were kindly authenticated by Professor Guangwan Hu, a senior taxonomist from the Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture (Wuhan Botanical Garden, Chinese Academy of Sciences). A voucher specimen was stored in the herbarium of the Key Laboratory.
For the sample preparation, the air-dried plant materials were firstly ground to a powder with a high-speed disintegrator. Then, an aliquot of 100 g accurately weighted plant powders was extracted ultrasonically three times using 60% ethanol at room temperature for 30 min to produce the crude R. prinoides extracts (CRE). Then the CRE were dispersed in water and extracted with petroleum ether (PE) and EA, successively. Finally, the EA extracts were loaded into a polyamide column, and the targeted semipurified R. prinoides extracts (SPRE) of the polyphenols enriched fraction was prepared by eluting with 80% ethanol solution. The yields of CRE and SPRE were referred to the percentage of the dry weight of the extracts to the dry plant samples, and calculated based on the formula below: Weight of dry extract Weight of dry plant samples × 100 (1)

Determination of Total Phenolic Content (TPC)
The total phenolic contents in the above two extracts were measured using the Folin-Ciocalteu method [39] with some modifications. In brief, 200 µL of the properly diluted sample solutions or the standard gallic acid (GA) solutions were firstly mixed with an equivalent Folin-Ciocalteu reagent (0.25 M) by vortexing. After that, 1000 µL of Na 2 CO 3 (1.0 M) and 600 µL of H 2 O were added and mixed gently. Later, the reaction mixtures were further cultivated for 1 h at room temperature in the dark, and finally, the absorbed optical density (AOD) was recorded at the wavelength of 760 nm. The GA was served as the standard, and the TPC was defined as milligram of GA equivalents per gram of the sample (mg GAE/g).

Determination of Total Flavonoid Content (TFC)
The total flavonoid content in all samples was determined with the previous colorimetric report [40] using rutin as a standard flavonoid compound. In brief, 180 µL of the properly diluted extracts or rutin standard solutions were firstly mixed with 1080 µL of H 2 O and subsequently with 60 µL of 5% NaNO 2 solution. After incubation for 6 min, 120 µL of 10% AlCl 3 solution was then added and left to stand for 6 min. After that, 360 µL of 4% NaOH solution was added, and these reaction mixtures were kept for another 15 min at room temperature. The AODs of these reaction mixtures were detected at the wavelength of 510 nm, and the TFC was defined as milligram of rutin equivalents per gram of the sample (mg RE/g).

DPPH Free Radical Scavenging Activity
The DPPH free radical scavenging assay was carried out in 96-well microliter plates according to a previously described method [41]. Briefly, 10 µL of the Trolox or sample solutions at various concentrations were mixed with 190 µL of the DPPH methanol solution (100 µM, final concentration) in the 96-well plate. Then, the reaction mixtures were shaken gently and cultivated in the dark for 0.5 h at room temperature. Thereafter, the discoloration of DPPH radicals was detected through recording the AOD at the wavelength of 517 nm with a Tecan microplate reader (Infinite M1000, Switzerland). BHT was used as the positive control. The DPPH radical scavenging activity (RSA) was calculated as the equation: RSA (%) = [(Acontrol − Asample)/Acontrol] × 100%. Each sample solution test was repeated three times, and all the results of the two flavonoid fractions were described to be micromolar Trolox equivalents (TE) per gram of the sample (µM TE/g).

ABTS Free Radical Scavenging Activity
The ABTS radical scavenging activity was assessed using a previous method [41]. In brief, the ABTS + stock solution was first prepared by mixing the ABTS solution (7.0 mM, in H 2 O) and an equal volume of the potassium persulfate solution (2.45 mM, in H 2 O) in darkness for 12-16 h. After that, the ABTS + working solution was prepared through successively diluting the ABTS + stock solution with 80% ethanol to the AOD of 0.70 ± 0.01 at the wavelength of 734 nm. The reaction mixture was composed of 10 µL of the Trolox or sample solution and 190 µL of the ABTS + working solution, and then cultivated in the dark for 0.5 h. The AOD at the wavelength of 734 nm was obtained in triplicate. The calculation and expression of ABTS were consistent with the above mentioned DPPH radical scavenging activity.

Measurement of LPS-stimulated NO in Macrophage RAW 264.7 Cells
The RAW264.7 macrophage cells were purchased from the American Type Culture Collection (ATCC). The cells were maintained at 37 • C in DMEM medium supplemented with the 10% FBS, 1% penicillin-streptomycin with 5% CO 2 in a humidified incubator. The nitrite concentration, an indicator of the NO synthesis, was measured based on the previously described Griess reaction method [42]. Briefly, RAW 264.7 cells were transferred into the 96-well plate with a density of 5 × 10 4 cells/well and kept for 24 h. These cells were incubated with various concentrations of the SPRE solution for 2 h and then followed by the incubation with 10 ng/mL of LPS for another 24 h. Meanwhile, 10.0 µg/mL of aspirin was used as the positive control. After that, 100 µL of the culture supernatant was cultivated with the same volume of Griess reagent for 15 min at room temperature, and the AOD was finally determined with a microplate reader at the wavelength of 540 nm, according to the manufacturer's instructions.

COX-2 Inhibition Assay
Along with the aspirin used as the positive control, the in vitro COX-2 inhibitory test was implemented based on our previous method [34]. Briefly, COX-2 (1U, 20 µL) was mixed with Tris-HCl (100 mM, 150 µL) and hematin (1.0 µM, 10 µL) and shaken gently for 2.0 min. After that, 10 µL of the tested sample solutions (0.46 − 333 µg/mL) was added and incubated for 5.0 min. Then, arachidonic acid (100 µM, 10 µL) and TMPD (10 µM, 10 µL) were added to the initiated the reaction, and finally, the reaction mixtures were terminated by the addition of HCl solution (2.0 M, 20 µL) after incubation for 5.0 min. The optical values (OD) of the reaction mixtures were monitored at the wavelength of 590 nm. The IC 50 value and its dose-dependent curve of the SPRE sample were acquired based on the non-linear regression analysis (GraphPad, v5.01). The data were expressed as Mean ± SD of three replicates.

HPLC-UV/ESI-MS/MS Analysis of R. prinoides
The HPLC-UV/ESI-MS/MS Analysis of R. prinoides was implemented by using the Termo Accela 600 HPLC system, which was connected with the TSQ Quantum Access MAX mass spectrometer (Termo Fisher Scientific, San Jose, CA, USA). A Waters SunFire™ RP-C18 column (150 mm × 4.6 mm, 3.5 µm) was employed for the chromatographic separation. The H 2 O (A) and acetonitrile (B) were used as the mobile phases, and the elution gradient was set as follows: 0-2 min, 15% B; 2-45 min, 14% − 45% B; 45-55 min, 45% − 60% B; 55-60 min, 60% − 15% B. The injection volume was 10 µL, the flow rate was 0.5 mL/min, the column temperature was kept at 30 • C, and the on-line UV chromatograms were monitored at 360 nm. The relative quantitative analysis was calculated by the proportion of the AUC of one component in the HPLC-UV chromatogram profile to the total AUC of all components. For the following ESI-MS/MS analysis, the MS/MS conditions were set the same as those of our previous study [23].

Statistical Analysis
All the statistical analysis was performed using the SPSS program (V 16.0). One-way ANOVA with the Tukey and LSD tests was employed to compare the significance between groups. All the values are present to be the Mean ± SD (standard deviation, n = 3), and the significant differences were considered at p ≤ 0.05 and p ≤ 0.01.

Conclusions
In the present study, the noteworthy antioxidant and anti-inflammatory activities of the polyphenols of R. prinoides from Mount Kenya were comprehensively evaluated for the first time. As a result, the polyphenol enriched extracts of CRE and SPRE displayed strong antioxidant activities by capturing the DPPH and ABTS + free radicals. Along with the higher contents of polyphenols and flavonoids, and the stronger antioxidant activities, the SPRE showed potential anti-inflammatory activities by reducing the NO production and the COX-2 activity. On the other hand, further HPLC-UV/ESI-MS/MS analysis of the polyphenol profile of R. prinoides revealed that flavonoids and their glycosides not only made up the major ingredients but were also potentially responsible for its strong antioxidant and anti-inflammatory activities. In conclusion, the present study comprehensively demonstrated that the high content of polyphenols in R. prinoides, along with noteworthy antioxidant and anti-inflammatory activities, confirmed that R. prinoides is a good natural source of polyphenols and flavonoids, and provided further scientific evidences for its use as folk medicine and with good potential for future healthcare practice.