Viburnum mullaha D. DON fruit (Indian cranberry): A potential source of polyphenol with rich antioxidant, anti-elastase, anti-collagenase, and anti-tyrosinase activities

ABSTRACT

Viburnum mullaha (Buch.-Ham. Ex D. Don), is an underexplored wild edible fruit of Indian Himalayan region, analyzed for total polyphenol contents, antioxidant, anti-elastase, anti-collagenase, and anti-tyrosinase activities using in vitro assays. High values of total phenolic contents of 1257 ± 40 mg gallic acid equivalents/100 g fruit weight and total flavonoid contents of 3501 ± 203 mg catechin equivalents/100 g fruit weight were observed. V. mullaha fruit extracts showed outstanding antioxidant activities (ABTS⁺, 1,1-diphenyl-2-picrylhydrazyl, superoxide anion, linoleate peroxyl radicals scavenging, and ferric reducing activities) and remarkable anti-elastase, anti-collagenase, and anti-tyrosinase activities. High resolution liquid chromatography–mass spectroscopy analysis revealed presence of 15 phenolic compounds, namely, chlorogenic acid, acetyl salicylic acid, dihydrorobinetin, dihydromyricetin, 2-isoprenylemodin, rutin, cosmosiin hexaacetate, pectolinarin, dihydroquercetin, eriodictyol, iriginol hexaacetate, theaflavin, epicatechin-pentaacetate, lomatin, and peucenin in fruit extracts. This study recommends utilization of V. mullaha fruit as functional food with prospective pharmaceutical, nutraceutical, and cosmeceutical properties.

KEYWORDS:

• Antioxidant
• Anti-elastase
• Anti-collagenase
• Anti-tyrosinase
• Polyphenols
• Wild-edible fruits of Himalaya

Introduction

Increased vegetable and fruit consumption has been known to be associated with lesser risk of a number of chronic diseases, such as coronary heart diseases, cancers, immune dysfunction, and diabetes mellitus.^[1] As a primary food source, vegetables and fruits provide health-promoting nutrients including vitamins, minerals, phenolics, and flavonoids. Fruits such as berries, bananas, apples, grapes, mangoes, pomegranates, papayas, oranges, pears, and pineapples have been shown to possess many health boosting properties including antioxidant, anti-cancer, anti-inflammatory, and antimicrobial due to their elevated phenolic and flavonoid contents.^[2–5] Besides commercial fruits, wild edible fruits have also gained worldwide concern due to their higher polyphenol contents and outstanding antioxidant activities.^[6–9]

Wild edible fruits contribute significantly to the nutrition of tribal and rural inhabitants of North West Himalaya.^[10] They have served as dietary staples and medicines for thousands of years.^[11] Limited studies on wild edible fruits of Uttarakhand of North West Himalaya have shown presence of abundant polyphenols and associated antioxidant, anti-microbial, anti-inflammatory, and anti-proliferative activities.^[12–17] Viburnum mullaha (Buch.-Ham. Ex D. Don), is one of the underutilized wild edible plants of Indian Himalayan region. The plant species grows abundantly in nature between 1500–3300 m amsl in Uttarakhand, Jammu, and Kashmir and North Bengal.^[18] Viburnum mullaha berries are reddish yellow in color and broadly oblong fruits, ripen in August–September.^[11,18] The berries are highly nutritious, delicious, and rich in minerals and vitamins.^[19] Despite this, nothing is known about the polyphenol contents and their bioactivities of neutraceutical and cosmeceutical importance including antioxidant, anti-skin aging, and skin whitening activities. Therefore, the present study was aimed to (1) assess the total phenolics and flavonoid contents of the fruit extracts of V. mullaha; (2) evaluate the extracts for antioxidant activities using various in vitro assays; (3) determine the anti-skin aging and skin whitening activities using in vitro anti-elastase, anti-collagenase, and anti-tyrosinase assays; and (4) explore polyphenolic composition of fruit extract by high-resolution liquid chromatography–mass spectroscopy (HR-LC-MS) analysis.

Materials and methods

All the chemicals were of analytical grade and more than 99% pure. 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitro blue tetrazolium (NBT), phenyl methosulfate (PMS), nicotinamide adenine dinucleotide (NADH), β-carotene, linoleic acid, ferrozine, catechin, kojic acid, N-succinyl–ala-ala-ala-p-nitroanilide (AAAPVN), N-[3-(2-furyl)acryloyl]-leu-gly-pro-ala (FALGPA), 3-4-dihydroxy-L-phenylalanine (L-DOPA), epigallocatechin gallate (EGCG), and Clostridium histolyticum collagenase (EC 3.4.24.3), porcine pancreatic elastase (EC 3.4.21.36), and mushroom tyrosinase (EC 1.14.18.1) were procured from Sigma-Aldrich (St Louis, MO, USA). 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) was obtained from Calbiochem, Merck Pvt Ltd (Darmstadt, Germany). Other reagents were purchased from HiMedia Pvt. Ltd. (Mumbai, India).

Collection, identification, and authentication of fruits

Ripened fruit samples (along with small twig containing leaves) were harvested from various locations of Chopta, District Chamoli, in Uttarakhand state of India during the month of January. Fresh fruits were brought to the laboratory and cleaned under running tap water and kept at −20°C until use within 1 month. The herbarium of fruit samples was authenticated by Prof R.D. Gaur, Department of Botany, H.N.B. Garhwal University, Srinagar, Uttarakhand.

Evaluation of total phenolic contents (TPCs) and total flavonoid contents (TFCs)

Fruit extracts were prepared with 80% aqueous acetone as solvent.^[13] The TPCs were determined by Folin–Ciocalteau method.^[20] Briefly, 1 mL fruit extracts was incubated with 2.5 mL Folin–Ciocalteau reagent (0.2N) at room temperature (RT) for 5 min. Thereafter, 2 mL of sodium carbonate solution (75 g/L in water), was added and further incubated for 2 h. Absorbance was measured using ultraviolet-visible (UV–Vis) spectrophotometer (Systronics, India, Model No. 119) at 760 nm against a water control. A standard calibration curve was plotted using gallic acid (0.2 mg/mL). The TPCs were expressed as mg gallic acid equivalent (GAE)/100 g fruit weight (FW).

TFCs of fruit extracts were determined according to Chang et al.^[21] There was 0.6 mL diluted extract incubated with 0.3 mL of sodium nitrite solution (5%), at RT for 5 min. Afterward, 0.6 mL aluminium trichloride solution (10%) was added and incubated further for 5 min. Absorbance was measured at 510 nm against water blank and TFC was expressed as mg catechin equivalents (CEs)/100 g FW using catechin (0.5 mg/mL) as standard.

Antioxidant activity

Antioxidant activities of fruit extracts were evaluated as free radical scavenging activities such as DPPH radical scavenging activity (DPPHRSA), ABTS⁺ radical scavenging activity (ABTSRSA), linoleate hydroperoxide radical scavenging activity (LPRSA), superoxide radicals scavenging activity (SORSA), ferrous metal chelating activity (FMCA) and ferric reducing activity (FRA) using the protocols mentioned by Saini et al.^[22]

Anti-elastase and anti-collagenase activity

Anti-elastase and anti-collagenase activities were determined according to Kim et al.^[23] for anti-collagenase assay; fruit extracts were pre-incubated with the collagenase enzyme (stock 0.8 U/mL) for 15 min before adding substrate to start the reaction. The final reaction mixture (150 μL) contained 50 mM Tricine buffer, 0.8 mM synthetic substrate FALGPA, 0.1 unit collagenase and various dilutions of fruit extracts. Absorbance at 335 nm was measured immediately after adding substrate and then continuously for 20 min using microplate reader (Fluostar Optima, BMG Labtech, Germany). Anti-collagenase activity of fruit extracts was determined as mg EGCG equivalents/100 g FW. EGCG (250 μM) was used as standard inhibitor.

Anti-elastase assay involved incubation of fruit extracts with the porcine pancreatic elastase (stock solution 3.33 mg/mL in sterile water) for 15 min before adding the substrate. The final reaction mixture (250 μL) contained 0.2 mM Tris-HCl buffer (pH 8.0), substrate 0.8 mM AAAPVN, 1 μg/mL elastase and 25 μL fruit extracts. Absorbance values between 381 and 402 nm (following pre-screen scans) were measured continuously for 20 min after adding substrate. EGCG (250 μM) was used as a standard and anti-elastase activity of the fruit extracts was represented as mg EGCG equivalents/100 g FW.

Anti-tyrosinase activity

Anti-tyrosinase activity was evaluated with respect to the fruit extracts ability to inhibit mushroom tyrosinase enzyme.^[24] Reaction mixture included 20 μL phosphate buffers (0.1 M pH 6.8), 20 μLL-DOPA (0.85 mM) as the substrate, and 20 μL diluted fruit extracts was incubated at 25°C for 10 min. Afterward, 20 μL mushroom tyrosinase (1000 U/mL) was added to initiate the reaction and incubated for 25 min. The absorbance was measured at 492 nm. Kojic acid (0.5 mg/mL) was used as positive control and anti-tyrosinase activity was measured as milligrams of kojic acid equivalents (KAE)/100 g FW.

HR-LC-MS

HR-LC-MS analysis of V. mullaha fruit extracts was performed at Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology, Mumbai, India on commercial basis. A small quantity of dried sample was dissolved with 80% acetone, and filtered through a glass syringe using 0.2 µm filter and then analysed in UHPLC (1290 Infinity Binary pump, Agilent Technologies) coupled with 6550 i-Funnel QTOF mass spectrometer (The mobile phase was a gradient of 0.1% formic acid in water and 0.1% formic acid in ACN at a flow rate of 0.3 mL/min for 30 min. The column used was Zorbax SB C18 Rapid Resolution HD, 2.1 × 100 mm, 1.8 µm (Agilent Technologies, USA). The nitrogen gas flow was 13 mL/min at 25°C, and the sheath gas flow was11 mL/min at 30°C with a nebulizer pressure at 35 psi. The capillary voltage was 3500 V with a nozzle voltage of 1000 V. Fragmentation energy was kept at 175 V. The analysis was done in both +ESI and –ESI mode, the data was acquired in Agilent Masshunter Data Acquisition software (Version B.05.00) and the data were analyzed in Agilent Masshunter Qualitative Software (Version B.06.00).

Statistical analysis

To rule out any inconsistencies, three independent extractions were performed and each extract was analyzed at least three times for each parameter. The results were expressed as mean of three independent experiments (n = 3) with calculation of standard error (SE). The statistical analysis of the data was performed using MS Excel and Prism 3 pad software (Microsoft, Redmond, WA, USA).

Results and discussion

TPC and TFC

The TPC was evaluated using gallic acid as standard (R² = 0.984). It was found to be 1257 ± 40 mg GAE/100 g FW in acetone extracts. TFC was determined using catechin as standard (R² = 0.999) and it was much higher (3503 ± 203 mg CE/100 g FW) than the TPC present in the extracts (Table 1). The study of TPC and TFC of other edible species of Viburnum genus excluding mullaha species have been carried out by several researchers.^[25–28] Among Viburnum sp, V. opulus was the most studied fruit. In an early research in V. opulus methanol fruit extracts, the TPC was found to be 132 ± 2.11 mg GAE/g dried fruit extracts.^[25] Further, Rop et al.^[26] showed that TPC and TFC ranged 6.8–8.29 g GAE and 3.14–4.9 g rutin equivalents/kg FW, respectively, in three different cultivars of V. opulus var. edule. Kraujalytė et al.^[27] studied TPC in six different V. opulus genotypes and it varied in the range of 5.4–10.6 mg GAE/g. Bhagat et al.^[28] determined TPC and TFC as 72.16 mg GAE and 110.49 mg quercetin equivalents/g in dried fruit extract of V. grandiflorum. The TPC and TFC values of V. mullaha fruit were comparable to the above mentioned species however much greater than some of the other wild edible fruits of the North-West Himalayan Region.^[17]

Table 1. TPC and TFC of V. mullaha fruit.

	TPC mg GAE/100 g FW	TFC mg CE/100 g FW
80% acetone extracts	1257 ± 40	3503 ± 203

Each value was expressed as mean ± SE (n = 3).

Antioxidant activities

Free radical scavenging activities of the V. mullaha fruit extracts were evaluated against varieties of free radicals including positive (ABTS⁺), neutral (DPPH), negative (superoxide), and lipid peroxides (linoleate peroxide; Table 2). The study revealed presence of outstanding free radical scavenging activities against all the tested free radicals. ABTSRSA was determined using BHA as a standard (R² = 0.981) and it was found to be 1095.2 ± 1.4 mg BHAE/100 g FW. DPPHRSA was determined by using ascorbic acid as standard (R² = 0.949). Observations showed presence of potent DPPHRSA in the fruit extracts (1428 ± 0.5 mg AAE/100 g FW). The linoleate hydroperoxide RSA was measured using BHA as a standard (R² = 0.931). The fruit extracts showed high inhibition activities (2107 ± 263 mg BHAE/100 g FW) against linoleate peroxyl radicals. V. mullaha fruit extracts showed excellent superoxide radicals scavenging activities (15016.14 ± 894 mg CE/100 g FW) as determined using catechin as standard (R² = 0.936). FRA activity was determined by using ascorbic acid as a standard (R² = 0.912). The fruit extracts demonstrated very high ferric reducing activities in the extracts (2176 ± 60 mg AAE/100 g FW). A low level of FMCA was also observed in the acetone extracts of fruit.

Table 2. Antioxidant activities of fruit extracts of V. mullaha.

ABTS mg BHAE/100 g FW	DPPH mgAAE/100 g FW	LPRSA mgBHAE/100 g FW	SORSA mgCE/100 g FW	FRAP mgAAE/100 g FW	FMCA mgEDTAE/100 g FW
1095.2 ± 1.4	1428.3 ± 0.5	2107 ± 263	15016 ± 894	2176 ± 60	14.3 ± 0.5

Each value was expressed as mean ± SE (n = 3).

V. mullaha fruit extracts demonstrated outstanding antioxidant activities with respect to all the tested in vitro assays except metal chelating activity (Table 2). Antioxidant activities of many species of Viburnum other than mullaha have been analyzed previously.^[25–29] Sagdic et al.^[25] demonstrated high antioxidant activities (315.50 ± 8.2 mg g^–1 in dried methanolic extracts using phosphomolybdenum complex method) in dried fruits of V. opulus. Altun et al.^[29] studied antioxidant activities in the fruit extracts of Viburnum opulus and Viburnum lantana growing in Turkey by DPPH and superoxide anion methods. They revealed presence of strong DPPH inhibition activities in V. opulus and V. lantana fruit extracts (IC50 values of 0.057 and 0.085 mg/mL, respectively); however, no superoxide anion inhibition was detected in any of the fruit extracts while comparing with tocoferol. Rop et al.^[26] showed presence of superior DPPH and ABTS scavenging activities in V. opulus fruit extracts in the range of 8.5–9.75 and 9.1–11.12 AAE g/kg fresh mass^[26] which was evidently similar to the activities that was reported in V. mullaha fruit extracts in the present study. Further, Bhagat et al.^[28] demonstrated presence of a potent DPPH inhibition (IC₅₀ value-294.5 µg/mL) and ferric reducing (IC₅₀ value-1217 µg/mL) activities in the fruit of V. grandiflorum. While comparing with the antioxidant activities of the other wild edible fruits of the Indian Himalayan Region, V. mullaha exhibited greater or comparable activities than thatwere reported in other fruits^{[13,14,17,30]} excluding L. latifolia and L. asiatica fruit extracts.^[17]

Anti-elastase, anti-collagenase, and anti-tyrosinase activities

Anti-elastase and anti-collagenase activities of the fruit phenolics were determined using EGCG as standard (R² = 0.991 and R² = 0.963, respectively)^[31] and represented as mg EGCG equivalents/100 g FW (Table 4). The inhibition reactions were carried out with an extract concentrations equivalent to 0.8–5.0 mg of FW/mL and 1.5–10 mg FW/mL. The fruit extracts significantly inhibited the elastase and collagenase enzymes (Fig. 1a and 1b). Inhibition was dose dependent, and the degree of inhibition was different for both the enzymes. Elastase was observed to be highly sensitive for V. mullaha fruit extracts showing maximum inhibition of more than 90% at 5 mg FW/mL (Fig. 1a). The median inhibitory concentration (IC₅₀) was found to be 1.4 mg FW/mL (Table 3). Collagenase enzyme was moderately sensitive to fruit extracts (little less sensitive than elastase enzyme) showed more than 60% inhibition at 10 mg FW/mL (Fig. 1b). The IC₅₀ was found to be 9.55 mg FW/mL (Table 3). The total anti-elastase and anti-tyrosinase activities of the V. mullaha fruit in term of EGCG were found to be 1123 ± 215 mg EGCGE/100 g FW and 1593 ± 128 mg EGCGE/100 g FW (Table 4). Anti-tyrosinase activity of the V. mullaha fruit extracts was assessed using kojic acid as standard inhibitor (R² = 0.937)^[23] and represented as mg KAE/100 g FW (Table 4). The anti-tyrosinase activity was analyzed at fruit extract concentration of 0.8 to 8.0 mg FW/mL. The anti-tyrosinase activity of fruit extract was dose dependent and maximum inhibition of 83% was observed at 8.0 mg FW/mL concentration (Fig. 1c). The IC₅₀ of the fruit extracts for tyrosinase inhibition was found to be 4.01 mg FW/mL (Table 3). The total anti-tyrosinase activity in V. mullaha fruit extracts was observed to be 10518 ± 140 mg KAE/100 g FW (Table 4).

Table 3. IC50 value for anti-elastase, anti-collagenase and anti-tyrosinase activities of Viburnum mullaha.

In vitro assay		IC50 values
		V. mullaha fruit extracts (mg FW/mL)	Positive control (µg/mL)
Anti-elastase assay		1.4	2.5
Anti-collagenase assay		9.55	18.8
Anti-tyrosinase assay		4.01	0.93

EGCG was used as positive control for the anti-elastase and anti-collagenase assay while kojic acid was used as positive control for anti-tyrosinase assay.

Table 4. Anti-elastase, anti-collagenase, and anti-tyrosinase activities of V. mullaha fruit extracts.

	Anti-elastase(mg EGCGE/100 g FW)	Anti-collagenase(mg EGCGE/100 g FW)	Anti-tyrosinase(mg KAE/100 g FW)
V. mullaha acetone	1123 ± 215	1593 ± 128	10518 ± 140

Each value was expressed as mean ± SE (n = 3).

Figure 1. Inhibition activities of V. mullaha fruit extracts. A: porcine pancreatic elastase; B: Clostridium histolyticum collagenase; C: mushroom tyrosinase. Extracts concentration was equivalent to the mg FW/mL reaction medium; each value is expressed as mean ± SE (n = 3).

To the best of our knowledge, present study is the first one to demonstrate anti-elastase and anti-collagenase activities of fruits of any of the species of Viburnum genus. Although, among the Viburnum genus, the dried leave extracts (500 µg/mL concentration) of Virburnum odoratissimum showed excellent anti-elastase activities exhibiting more than 85% inhibition while leave extracts of two other species namely V. dilatatum and V. erosum showed 48% and 43% inhibition of elastase, respectively.^[35] To date, no study is available on the anti-skin aging potentials of the fruits of any of the species of Viburnum genus. Anti-elastase and anti-collagenase activities of the polyphenols are considered as their anti-skin aging potentials.^[36] Many plant extracts (including fruit) have been earlier shown to possess anti-elastase and anti-collagenase activities as their anti-skin aging properties.^{[17,31,33–35]} Anti-tyrosinase activities of polyphenols have been widely reported.^[37] A recent study by Yilmaz et al.^[38] has testified presence of potent anti-tyrosinase activity in methanolic extracts of V. opulus fruits. However, we have not come across any report on anti-tyrosinase activity of the fruits of Viburnum mullaha. The tyrosinase inhibitory activity of V. mullaha fruits in the present study was comparable to the other wild edible fruits of Indian Himalayan Region.^[17]

Polyphenolic composition

Composition of phenolic compounds in aqueous acetone fruit extracts of V. mullaha was determined qualitatively by HR-LC-MS analysis. The identification of compounds was performed by comparing their mass spectra and fragmentation pattern with the polyphenol database (supplied by Agilent Technologies) using Agilent Masshunter Qualitative Software (Version B.06.00). The analysis revealed presence of 15 phenolic compounds including two phenolic acids (chlorogenic acid, acetyl salicylic acid) and 13 flavonoids (dihydrorobinetin, dihydromyricetin, 2-isoprenylemodin, rutin, cosmosiin hexaacetate, pectolinarin, dihydroquercetin, eriodictyol, iriginol hexaacetate, theaflavin, epicatechin-pentaacetate, lomatin and peucenin; Table 5). The structures of the compounds are presented in the Fig. 2. It was evident that V. mullaha fruit is a rich source of polyphenols which validated the high antioxidant, anti-elastase, anti-collagenase and anti-tyrosinase activities of the fruit extracts. Our findings are supported by the facts that some of the identified phenolic compounds namely chlorogenic acid, dihydromyricetin, rutin, dihydroquercetin, theaflavin, and epicatechin have already been shown to possess anti-skin aging and skin-lightening activities.^{[36,37,39,40–43]} The present study is the first to report polyphenolic composition of V. mullaha fruit and also first one to demonstrate presence of acetyl salicylic acids, dihydrorobinetin, dihydromyricetin, 2-isoprenylemodin, cosmosiin (apigenin 7-glucoside) hexaacetate, pectolinarin, eriodictyol, iriginol hexaacetate, theaflavin, lomatin, and peucenin in any of berry species of the Viburnum genus. Earlier, polyphenolic contents have been analyzed in berries of other species of Viburnum including V. dilatatum, V. opulus, and V. sargentii.^{[27,32,44–47]} Kim et al.^[32] reported phenolic composition of squeezed fruit juice extracts of Viburnum dilatatum by employing NMR and liquid chromatography/MS techniques. The phenolic compounds included cyanidin 3-sambubioside, cyanidin 3-glucoside, 5-O-caffeoyl-4-methoxyl quinic acid, quercetin, and chlorogenic acid.^[32] Veliglu et al.^[44] demonstrated chlorogenic acid as the major phenolics (54% of total phenolics), in addition to (+)-catechin, (-)-epicatechin, cyanidin-3-glucoside, cyanidin-3-rutinoside, and six different glucosides of quercetin in the fruit juice of European cranberry bush (Viburnum opulus L.) using high-performance liquid chromatography. Ozrenk et al.^[45] showed presence of gallic acid, catechin, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, o-coumaric acid, protocatechuic acid, vanillic acid, rutin, and quercetin in fruit juice of Viburnum opulus, in which catechin was the major flavonoids (284.96 mg kg⁻¹–352.04 mg kg⁻¹ FW) as measured by HPLC. Chirigiu et al.^[46] further confirmed presence of caffeic acid, p-coumaric acid, ferulic acid, 3,5, dimethoxy-4-hydroxycinnamic acid, rutin, quercitol, and kaempferol in V. opulus. Kraujalyte et al.^[27] reported presence of nine phenolic compounds (chlorogenic acid, neochlorogenic acid, quinic acid, catechin, epicatechin, procyanidin C1, and three other unidentified compounds) in V. opulus juice by using ultra high performance liquid chromatography coupled to quadruple and time-of-flight mass spectrometry (UPLC-QTOF-MS). Xie et al.^[47] isolated seven phenolic compounds from the fruits of V. sargentii Koehne by silica gel column chromatography and preparative HPLC. The phenolic compounds were identified as (−)-epicatechin 5,7,4′-trihydroxy-flavonoid-8-C-β-D-glucopyranoside, 1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-α-L-rhamnopyranoxypropyl)-2-methoxyphenoxy]-1,3-propane-diol (erythro), 1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-α-L-rhamnopyranoxypropyl)-2-methoxyphenoxy]-1,3-propanediol(threo), (R)-4-hydroxylphenol O-(6-O-oleuropeoyl)-β-D-glucopyranoside, (R)-3-methoxy-4-hydroxylphenol O-(6-O-oleuropeoyl)-β-D-glucopyranoside, quercetin-3-O-rutinoside.^[47]

Table 5. Polyphenolic composition of the acetone fruit extracts of V. mullaha.

S. No	Compounds	Retention time (min)	MS (m/z)	Mass	Formula
1.	Acetyl salicylic acid	7.137	163.0378	180.0412	C9 H8 O4
2.	Chlorogenic acid	7.138	355.0994	354.092	C16 H18 O9
3.	Dihydroquercetin	0.941	287.0529	304.0561	C15 H12 O7
4.	Dihydrorobinetin	7.72	287.0527	304.056	C15 H12 O7
5.	Dihydromyricetin	9.194	303.0475	320.0508	C15 H12 O8
6.	2-Isoprenylemodin	26.0	343.0945	338.1158	C20 H18 O5
7.	Rutin	8.95	633.1368	610.1474	C27 H30 O16
8.	Cosmosiin hexaacetate	55.26	684.1965	684.1707	C33 H32 O16
9.	Pectolinarin	7.73	661.1683	622.2049	C29 H34 O15
10.	Eriodictyol	40.049	309.0119	288.0584	C15 H12 O6
11.	Iriginol hexaacetate	25.075	571.1184	570.1109	C27 H22 O14
12.	Theaflavin	24.829	569.1024	564.1236	C29 H24 O12
13.	Epicatechin-pentaacetate	17.076	539.0928	500.13	C25 H24 O11
14.	Lomatin	27.78	229.0842	246.0875	C14 H14 O4
15.	Peucenin	20.857	243.0996	260.1028	C15 H16 O4

Figure 2. Polyphenols in the 80% acetone fruit extracts of V. mullaha.

Conclusion

Present study is the first exhaustive analysis of total polyphenol contents, antioxidant, anti-elastase, anti-collagenase, anti-tyrosinase activities, and phenolic composition of the fruit extracts of Viburnum mullaha, a wild edible fruit of North-West Himalayan Region of India. The study revealed that V. mullaha fruit extracts possess elevated phenolic and flavonoid contents with outstanding antioxidant activities and excellent inhibitory activities against enzymes of skin aging and melanin biosynthetic pathway. Study also showed presence of a large number of polyphenols with known anti-oxidant and anti-aging properties. These in vitro antioxidant, anti-skin aging and skin lightening activities of V. mullaha fruit extracts are highly appreciable suggesting its utilization for the development of safe and effective formulations for general health maintenance and effective anti-skin aging, skin whitening cosmetics.

Funding

The authors are grateful for financial support of this study which was provided in part by the Uttarakhand State Biotechnology Programme, Uttarakhand, India (USBD 06/Guard-7/K.Dangwal/R&D Project/Rishikesh/2012) and Modern Institute of Technology, Rishikesh, Uttarakhand, India.

Declaration of conflict

The authors declare there is no conflict of interest.

Acknowledgments

We are highly obliged to Dr. Partha Roy, Department of Biotechnology, Indian Institute of Technology, Roorkee, Uttarakhand, India, for providing instrumentation facility for anti-elastase, anti-collagenase, and anti-tyrosinase activity analysis. Authors also acknowledge the SAIF, Indian Institute of Technology, Mumbai, for the timely completions of HR-LC-MS analysis of fruit extracts samples.

Additional information

Funding

The authors are grateful for financial support of this study which was provided in part by the Uttarakhand State Biotechnology Programme, Uttarakhand, India (USBD 06/Guard-7/K.Dangwal/R&D Project/Rishikesh/2012) and Modern Institute of Technology, Rishikesh, Uttarakhand, India.