Development of an Enzyme-Linked Immunosorbent Assayfor Specific Detection of Mulberroside A in Mulberry (Morusalba L.) Using Anti-mulberroside A Polyclonal Antibody

Jukrapun Komaikul & Tharita Kitisripanya &

Hiroyuki Tanaka & Boonchoo Sritularak &

Waraporn Putalun

Received: 24 January 2013 /Accepted: 1 March 2013# Springer Science+Business Media New York 2013

Abstract An enzyme-linked immunosorbent assay(ELISA) was developed using polyclonal antibody (PAb)against mulberroside A (MuA), a major active componentfound in the root bark of mulberry (Morus alba L.). MuAwas conjugated with the carrier protein bovine serum albu-min for immunization to rabbits. The results showed that theantibodies were specific only for MuA and have very lowspecificity for its aglycone, oxyresveratrol. The ELISA as-say was suitable for quantitating MuA in the range of 0.17–15.62 μg/mL with a relative standard deviation (RSD) ofless than 5 % for both intra- and inter-assay precision levels.The recovery rates of MuA in the samples were in the rangeof 97.6–101.4 % with a RSD of less than 5 %. The devel-oped ELISA exhibited a good correlation value (R2=0.994)with the standard high-performance liquid chromatographymethod in the crude extracts of plant samples. These resultssuggest that the developed ELISA method using PAb

against MuA can be applied to determine MuA content withhigh specificity, rapidity, and simplicity in mulberry sam-ples. The developed ELISA method described could proveto be useful as an analytical tool for quality control ofmulberry and their products.

Keywords Mulberroside A .Mulberry (Morus alba L.) .

Enzyme-linked immunosorbent assay . Polyclonal antibody

Introduction

Mulberry (Morus alba L.) is commonly cultivated in Thailandand Asian countries for various purposes, including the use ofleaves as a food for silkworm, ripe fruit as food supplement, andtwig and root bark as traditional medicine (Song et al. 2009).Recently, mulberry root bark, twig, and leaves have beeninvestigated for use as natural health products and functionalfood due to their health benefits (Kumar and Chauhan 2008).Mulberry root bark has been used as an antitussive, anti-inflammation, and anti-asthmatic agent (Piao et al. 2010).Mulberroside A (MuA; Fig. 1) is a major stilbene glycosidefrom the root bark of mulberry. MuA has been identified as theactive compound in mulberry associated with potent uricosuricand nephroprotective effects (Wang et al. 2011) and anti-tyrosinase activity (Park et al. 2011). MuA can be convertedby the intestinal microbes or directly by glycosidase to itsaglycone part, oxyresveratrol (Kim et al. 2010). Oxyresveratrolshows many pharmacological effects including anti-tyrosinaseactivity (Tengamnuay et al. 2006), anti-viral activity (Galindo etal. 2011; Lipipun et al. 2011), antioxidant activity (Aftab et al.2010), hepatoprotective effect (Shi et al. 2008), and

J. Komaikul : T. Kitisripanya :W. Putalun (*)Faculty of Pharmaceutical Sciences, Khon Kaen University, KhonKaen 40002, Thailande-mail: waraporn@kku.ac.th

J. Komaikul : T. Kitisripanya :W. PutalunResearch Group for Pharmaceutical Activities of Natural Productsusing Pharmaceutical Biotechnology (PANPB), National ResearchUniversity-Khon Kaen University, Khon Kaen 40002, Thailand

H. TanakaGraduate School of Pharmaceutical Sciences, Kyushu University,Fukuoka 812-8582, Japan

B. SritularakFaculty of Pharmaceutical Sciences, Chulalongkorn University,Bangkok 10330, Thailand

Food Anal. MethodsDOI 10.1007/s12161-013-9598-4

neuroprotective effect (Horn et al. 2004). To date, natural healthproducts and cosmetics containing mulberry root extracts arewidely available in the market. The content of MuA in mulber-ry varies depending on the cultivation condition and the sourceof plants. Besides, it is difficult to determine the active com-pound in various types of samples with different interferencesand a large number of samples. High-throughput analysis isrequired for the determination of active compound in a largenumber of samples as well as different types of samples withoutsample pretreatment (Yusakul et al. 2012). Therefore, a reliable,sensitive, and high-throughput analysis method is necessary forquality control of the plant raw material and final product.

High-performance liquid chromatography (HPLC)methods were developed for stilbene glycoside analysis inplant samples (Piao et al. 2010) and biological fluids (Mei etal. 2012). The chromatographic techniques require compli-cated and expensive equipment and are not appropriate forhigh-throughput analysis because they are time-consumingand the samples may interfere with other compounds.Enzyme-linked immunosorbent assay using an antibody isa rapid and economical method for the determination ofbioactive compounds from medicinal plants such asmangiferin (Yusakul et al. 2012), aculeatiside A (Putalunet al. 2002), daidzin (Pongkitwitoon et al. 2010), puerarin(Pongkitwitoon et al. 2011), and asiaticoside (Tassanawat etal. 2012). The produced antibody interacted with high spec-ificity to the compound of interest; therefore, the immuno-assay can be applied for detection of a compound in varioustypes of samples. However, an antibody-based immunoas-say to determine MuA content has not been reported to date.In the present paper, we produced and characterized PAbagainst MuA for the specific determination of MuA inmulberry samples. The method development, validation,and application are presented in this paper.

Experimental

Chemicals

MuA was isolated from the root bark of M. alba asdescribed previously (Hirakura et al. 1986). The

structure was confirmed by comparing nuclear magneticresonance data in the literature. Oxyresveratrol wasobtained from Hangzhou Great Forest Biomedical(Hangzhou, China). Bovine serum albumin (BSA), ov-albumin (OVA), and Freund's complete and incompleteadjuvants were purchased from Sigma (MO, USA).Peroxidase-labeled anti-rabbit IgG was purchased fromMP Biomedicals (Ohio, USA). 2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was pur-chased from Wako (Osaka, Japan). All other chemicalswere standard analytical reagent-grade commercialproducts.

Plant Materials and Sample Preparation

Mulberry (M. alba) samples were collected from the Facultyof Pharmaceutical Sciences, Khon Kaen University, Thai-land (April 2011) and the Graduate School of Pharmaceuti-cal Sciences, Kyushu University, Japan (April 2012). Thereference specimens (NI-PSKKU 062–063) were depositedat the Herbarium of the Faculty of Pharmaceutical Sciencesat Khon Kaen University in Thailand.

Cell suspension culture was obtained from the leaf-derivedcallus of M. alba cultured on a Murashige and Skoog (MS)liquid medium supplemented with 0.1 mg/L thiadiazuron and1 mg/L naphthaline acetic acid (NAA). Root culture wasobtained from the root of M. alba cultured on a half-strengthMS liquid medium supplemented with 1 mg/L NAA.

The dried powder of plant samples (10 mg) was weight-ed, extracted with methanol (500 μL), and then sonicatedfor 15 min. The extracts were centrifuged at 3,000 rpm for3 min to collect the supernatant. This extraction procedurewas repeated three times. The combined extract was evap-orated at 50 °C and re-dissolved in methanol (1 mL). Con-sequently, sample solutions were diluted into anappropriated concentration for MuA determination byELISA and HPLC.

Synthesis of MuA Conjugates

The MuA–BSA conjugate was synthesized using theperiodate oxidation method (Tassanawat et al. 2012). MuA

HO

OH

O

O

OO

OH

OHHO

HO

HO

HO

OH

OHHO

OH

O

O

OO

OH

OHHO

HO

HO

HO

OH

OH

Fig. 1 Structure ofmulberroside A

Food Anal. Methods

(4 mg) in 0.4 mL dimethyl sulfoxide was slowly added to0.6 mL of aqueous solution containing sodium periodate(4 mg) and then stirred at room temperature for 1 h. Next,BSA (5 mg) in 1 mL carbonate buffer (50 mM, pH9.6) wasadded and stirred for 5 h. The reaction mixture was dialyzedwith water (five times), then lyophilized. Finally, MuA–BSA (3.6 mg) was obtained. The same method was usedto synthesize MuA–OVA (4.2 mg).

Determination for the Hapten Number of MuA–BSA

The hapten number in the MuA–BSA conjugate was deter-mined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) similarly aspreviously described (Mei et al. 2012). The MuA–BSAconjugate (1–10 pmol) was mixed with a 10−3-fold molarexcess of saturated sinapinic acid in an aqueous solutioncontaining 30 % of acetonitrile and 0.15 % trifluoroaceticacid. The supernatant of the mixture was subjected toMALDI-TOF-MS (Autoflex III Smartbeam, Bruker).

Immunization

Female New Zealand white rabbits (10 weeks old, 2.5 kg)were obtained from the National Animal Centre, MahidolUniversity, Nakhon Pathom, Thailand. All institutional andnational guidelines for the care and use of laboratory ani-mals were followed. Animal handing and the treatmentprotocol were conducted according to Khon KaenUniversity's Guidelines for the Care and Use of LaboratoryAnimals and were approved by the Animal Ethics Commit-tee of Khon Kaen University (record no. AEKKU 44/2555).The rabbits were immunized subcutaneously with 1 mL 1:1emulsion mixture of MuA–BSA (0.6 mg) in phosphate-buffered saline (PBS) and Freund's complete adjuvant. After4 weeks, the second immunization was performed using thesame conjugate emulsified with Freund's incomplete adju-vant (intramuscular), and final immunization was carried outwith the same emulsion mixture 2 weeks later. Seven daysafter the third immunization, rabbit serum was collected andstored at −20 °C until use. The serum was purified usingprotein G FF column.

Indirect ELISA

A 96-well immunoplate (Maxisorb Nunc, Roskilde, Den-mark) was coated with 1 μg/mL MuA–OVA in carbonatebuffer (100 μL/well) and then treated with 300 μL PBScontaining 1 % of gelatin for 1 h to reduce non-specificadsorption. The plate was washed three times with PBScontaining of 0.05 % Tween 20 (TPBS), then reacted with100 μL PAb against MuA for 1 h. The plate was washedthree times with TPBS and then incubated with 100 μL of a

1,000-fold dilution of peroxidase-labeled anti-rabbit IgG for1 h. After washing the plate three times with TPBS, 100 μLof substrate solution (0.1 M citrate buffer (pH4.0)containing 0.003 % H2O2 and 0.3 mg/mL ABTS) was addedto each well and incubated for 15 min. The absorbance at405 nm was measured using a microplate reader (model 550Microplate Reader BioRad Laboratories, CA, USA). Allreactions were incubated at 37 °C.

Indirect Competitive ELISA

After coating the 96-well immunoplate with MuA–OVA, itwas then treated with PBS containing 1 % of gelatin toreduce non-specific adsorption. Various concentrations ofMuA or samples (50 μL) dissolved with 20 % methanolwere incubated with 50 μL of PAb solution for 1 h. Theplate was washed three times with TPBS and then incubatedwith 100 μL of 1,000-fold dilution of peroxidase-labeledanti-rabbit IgG solution for 1 h. After washing the platethree times with TPBS, 100 μL of substrate solution wasadded to each well and incubated for 15 min. The absor-bance was measured using a microplate reader at 405 nm.

Method Validation

The reactivity of PAb was determined by indirect ELISA.The ELISA method was validated for sensitivity, precision,specificity, and accuracy. The indirect competitive ELISAwas performed to determine the competition when usingdifferent concentrations of MuA to evaluate the sensitivityand measurement range. Intra-assay precision was evaluatedby the variation of the measurement of standard MuA fromwell to well (n=5), and inter-assay precision was obtainedfrom the variation from different plates (n=5) expressed asrelative standard deviation (RSD%). The specificity of theassay was evaluated by a cross-reactivity experiment of PAbagainst various structure-related compounds. The accuracyof the developed ELISA method was evaluated by a recov-ery experiment using the analysis of MuA in the extract ofmulberry root bark to which the known amount of MuAwasadded. Briefly, the MuA standard solution with variousconcentrations (0–150 μg/mL) was spiked to the extract ofmulberry root bark and then analyzed by the developedindirect competitive ELISA. Each concentration was exam-ined in triplicate.

HPLC Analysis

The HPLC analysis was performed using a PerkinElmerSeries 200 LC pump with a PerkinElmer 785A UV/VISdetector. RP-18 column (LiChroCART®, 125×4 mm,5-μm particle size; Merck, Germany) was used as stationaryphase. The mobile phase consisted of 18 % aqueous

Food Anal. Methods

methanol containing 0.1 % acetic acid with a flow rate of1 mL/min. The detector wavelength was 320 nm (Piao et al.2010). Each sample was examined in triplicate.

Results and Discussion

Analysis of the MuA–BSA conjugate by MALDI-TOF-MSwas performed. The MALDI-TOF-MS spectrum of theMuA–BSA conjugate appeared approximately at m/z80,444 (Fig. 2). Molecular weights of BSA and MuA were66,382 and 508, respectively. The hapten number of MuA–BSA is approximately 25. Therefore, this conjugate wassufficient for immunization antibody.

After examining the reactivity of the antibody by addingvarious concentrations of anti-MuA PAb to each well of a96-well immunoplate and determining by indirect ELISA(Fig. 3), 2 μg/mL IgG was chosen for indirect competitiveELISA. The measurement range of MuA determined byindirect competitive ELISA was 0.17–15.62 μg/mL(Fig. 4).

The specificity of the assay was evaluated by exam-ining cross-reactivities (CRs) of PAb with indirect com-petitive ELISA and calculated using the method ofWeiler and Zenk (Weiler and Zenk 1976). Table 1shows that anti-MuA PAb did not have any cross-reaction with structure-related compounds includingoxyresveratrol, the aglycone part of MuA. The CRresult suggests that the anti-MuA PAb was highly spe-cific for MuA. These results indicate that the sugar partsare essential for reactivity and specificity of the anti-MuA PAb. Therefore, we developed the ELISA methodfor determination of MuA in mulberry.

Intra-assay and inter-assay precisions were determined.Table 2 shows that the maximum RSD of intra-assay was4.92 %, while that of inter-assay was 4.85 %. The factorsthat affect variations of the ELISA method might be due totemperature during incubation and variation in preparationreagents. Therefore, a standard curve should be generated inevery assay to reduce variations.

8044480444Fig. 2 The MALDI-TOF massspectrum of the MuA–BSAconjugate

Concentration of PAb ( g1000010010.010.0001

2.5

2.0

1.5

1.0

0.5

0.0

Abs

orba

nce

at 4

05 n

m

Concentration of PAb ( g/mL)1000010010.010.0001

2.5

2.0

1.5

1.0

0.5

0.0

µ

Fig. 3 Reactivities of PAb against mulberroside A

00.10.20.30.40.50.60.70.80.9

1

0.01 0.1 1 10 100 1000Concentration of mulberroside A (µg/mL)

Abs

orba

nce

at 4

05 n

m

y = -0.1505Ln(x) + 0.5855R2 = 0.9967

0.00.20.40.60.81.0

0 1 10 1000.1

Fig. 4 Standard curve for the determination of mulberroside A byELISA

Food Anal. Methods

Recovery experiment was studied to evaluate the accura-cy. After spiking solutions with various concentrations ofMuA into the mulberry sample, the sample extracts wereprepared, and the content of MuA in each sample wasdetermined by the developed ELISA method. As shown inTable 3, the percentages of recovery in the range of 97.6–101.4 % with a maximum RSD of 4.13 % indicated highextractability and recovery which were obtained from thedeveloped method.

In order to determine the correlation between theELISA and HPLC results, various samples of mulberrywere determined using the developed method. Table 4shows the results from MuA contents of mulberry indifferent samples. The concentrations of MuA deter-mined by ELISA had a good correlation with thosedetermined by HPLC. The correlation between the resultfrom the ELISA and HPLC had a high coefficient ofdetermination (r2=0.994). The root bark of mulberryshowed the highest amount of MuA, followed by theroot and fibrous root, respectively (Table 4). MuA wasdetected in the twig of the mulberry sample from Khon

Kaen Province using ELISA method, whereas HPLCmethod could not detect it due to a lower limit ofdetection. The developed ELISA method was also ap-plied for MuA detection in the in vitro culture ofmulberry. MuA contents in root and cell suspensioncultures were 19.34±0.53 and 10.31±0.76 mg/g drywt., respectively. This result shows that the developedELISA method is significantly more sensitive than theHPLC method. In addition, MuA was not found in theleaf of mulberry using both assays, suggesting a varia-tion of MuA accumulation from different parts of theplant. From these results, the developed ELISA methodwas applicable for detection of MuA in plant samplesand in vitro cultures of mulberry. To compare ELISAand HPLC techniques, the ELISA had fourfold highersensitivity than the HPLC as reported previously (Piaoet al. 2010). The prior advantages of the developed

Table 1 Cross-reactiv-ities of anti-mulberroside A PAb

Compound Cross-reactivity (%)

Mulberroside A 100

Oxyresveratrol <0.01

Resveratrol <0.01

Moscatilin <0.01

Artocarpin <0.01

Morusin <0.01

Norartocarpetin <0.01

Norcycloartocarpin <0.01

Isocyclomorusin <0.01

Cycloartobiloxanthone <0.01

Rutin <0.01

Glycyrrhizin <0.01

Artimisinin <0.01

Table 2 Intra- and inter-assay precisions of mulberroside A analysisby ELISA using anti-mulberroside A PAb (n=5)

Mulberroside A concentration(μg/mL)

Intra-assay RSD(%)

Inter-assay RSD(%)

0.20 3.01 3.14

0.39 2.07 3.34

0.78 3.11 2.79

1.56 2.88 3.02

3.13 3.31 4.71

6.25 4.58 4.85

12.5 4.92 4.84

Table 3 Recovery of mulberroside A determined by ELISA (n=3)

Concentration ofmulberroside A added(μg/mL)

Mulberroside Acontent (μg/mL)±SD

Recovery(%)

RSD(%)

0 206.41±7.36 – 3.56

18.75 228.27±9.42 101.4 4.13

37.50 242.46±3.87 99.4 1.59

75 283.07±10.07 100.3 3.59

150 352.95±10.28 97.6 3.96

Table 4 Determination of mulberroside A in mulberry using ELISAand HPLC

Mulberry sample Concentration of mulberroside A (mg/g drywt.)

HPLC ELISA

Khon Kaen, Thailand

Leaf ND ND

Twig ND 0.29±0.10

Fibrous root 5.24±0.39 5.32±0.37

Root 21.44±1.69 22.39±1.06

Root bark 24.32±0.20 26.86±2.69

Fukuoka, Japan

Leaf ND ND

Twig ND ND

Fibrous root 12.17±0.16 12.40±1.40

Root 14.34±1.14 14.60±0.85

Root bark 20.88±0.76 21.05±0.38

In vitro culture

Cell suspension culture 11.06±0.32 10.31±0.76

Root culture 18.47±2.11 19.34±0.53

ND not detected

Food Anal. Methods

ELISA system over traditional analytical methods are itssimplicity, rapidity, and sensitivity. Therefore, this vali-dated method is reliable for MuA determination in var-ious MuA-containing plant materials and final products.

Conclusion

The anti-MuA PAb was produced and characterized. The mea-surement range of the ELISA assay for PAb against MuAwas0.17–15.62 μg/mL, and it is highly specific for MuA. Thedeveloped ELISAwas applied for determination of MuA con-tents in mulberry samples. The contents of MuA from ELISAand HPLC were compared. The correlation between the twomethods had a coefficient of determination (r2) of 0.994. Insummary, our developed immunoassay using the anti-MuAPAb is a simple, rapid, sensitive, and cost-effective assay toevaluate the quality control of MuA in mulberry.

Acknowledgments This work was supported in part by the KanaeFoundation for the Promotion of Medical Science, Japan; the Facultyof Pharmaceutical Science, Khon Kaen University and the GraduateSchool, Khon Kaen University, Thailand.

Conflict of Interest Jukrapun Komaikul has no conflict of interest.Tharita Kitisripanya has no conflict of interest. Hiroyuki Tanaka has noconflict of interest. Boonchoo Sritularak has no conflict of interest.Waraporn Putalun has no conflict of interest. All institutional and nationalguidelines for the care and use of laboratory animals were followed.

References

Aftab N, Likhitwitayawuid K, Vieira A (2010) Comparative antioxi-dant activities and synergism of resveratrol and oxyresveratrol.Nat Prod Res 24:1726

Galindo I, Hernáez B, Berná J, Fenoll J, Cenis JL, Escribano JM,Alonso C (2011) Comparative inhibitory activity of the stilbenesresveratrol and oxyresveratrol on African swine fever virus repli-cation. Antivir Res 91:57

Hirakura K, Fujimoto Y, Fukai T, Nomura T (1986) Two phenolicglycosides from the root bark of the cultivated mulberry tree(Morus lhou). J Nat Prod 49:218

Horn TF, Andrabi SA, Spina MG, Lorenz P, Ebmeyer U, Wolf G(2004) Oxyresveratrol (trans-2,3′,4,5′- tetrahydroxystilbene) isneuroprotective and inhibits the apoptotic cell death in transientcerebral ischemia. Brain Res 1017:98

Kim JK, Kim M, Cho SG, Kim MK, Kim SW, Lim YH (2010)Biotransformation of mulberroside A from Morus alba results in

enhancement of tyrosinase inhibition. J Ind Microbiol Biotechnol37:631

Kumar RV, Chauhan S (2008) Mulberry: life enhancer. J Med PlantsRes 2:271

Lipipun V, Sasivimolphan P, Yoshida Y, Daikoku T, Sritularak B,Ritthidej G, Likhitwitayawuid K, Pramyothin P, Hattori M,Shiraki K (2011) Topical cream-based oxyresveratrol in the treat-ment of cutaneous HSV-1 infection in mice. Antivir Res 91:154

Mei M, Ruan JQ, Wu WJ, Zhou RN, Zhao HY, Yan R, Wang YT(2012) In vitro pharmacokinetic characterization of mulberrosideA, the main polyhydroxylated stilbene in mulberry Morus alba L.and its bacterial metabolite oxyresveratrol in traditional oral use. JAgr Food Chem 60:2299

Park KT, Kim JK, Hwang D, Yoo Y, Lim YH (2011) Inhibitory effectof mulberroside A and its derivatives on melanogenesis inducedby ultraviolet B irradiation. Food Chem Toxicol 49:3038

Piao SJ, Chen LX, Kang N, Qiu F (2010) Simultaneous determinationof five characteristic stilbene glycosides in root bark of Morusalbus L. (Cortex Mori) using high- performance liquid chroma-tography. Phytochem Anal 22:230

Pongkitwitoon B, Sakamoto S, Tanaka H, Tsuchihashi R, Kinjo J,Morimoto S, Putalun W (2010) Enzyme-linked immunosorbentassay for total isoflavonoids in Pueraria candollei using anti-puerarin and anti-daidzin polyclonal antibodies. Planta Med76:831

Pongkitwitoon B, Sakamoto S, Tanaka H, Tsuchihashi R, Kinjo J,Morimoto S, Putalun W (2011) Development of an enzyme-linked immunosorbent assay to determine puerarin and its agly-cone daidzein. J Nat Med 65:31

Putalun W, Tanaka H, Muranaka T, Shoyama Y (2002) Determinationof aculeatisides based on immunoassay using a polyclonal anti-body against aculeatisides A. Analyst 127:1328

Shi L, Zhang Z, Jin J (2008) Protective function of cis-mulberroside Aand oxyresveratrol from Ramulus mori against ethanol-inducedhepatic damage. Environ Toxicol Pharmacol 26:325

Song W, Wang HJ, Bucheli P, Zhang PF, Wei DZ, Lu YH (2009)Phytochemical profiles of different mulberry (Morus sp.) speciesfrom China. J Agr Food Chem 57:9133

Tassanawat P, Putalun W, Komaikul J, Sritularak B, JuengwatanatrakulT, Tanaka H (2012) Development of anti-asiaticoside polyclonalantibody-based immunoassay and applications for Centellaasiatica products and human serum. Food Anal Method 6:1320

Tengamnuay P, Pengrungruangwong K, Pheansri I, LikhitwitayawuidK (2006) Artocarpus lakoocha heartwood extract as a novelcosmetic ingredient: evaluation of the in vitro anti-tyrosinaseand in vivo skin whitening activities. Int J Cosmet Sci 28:269

Wang CP, Wang Y, Wang X, Ye JF, Hu LS, Kong LD (2011)Mulberroside a possesses potent uricosuric and nephroprotectiveeffects in hyperuricemic mice. Planta Med 77:786

Weiler W, Zenk H (1976) Radioimmunoassay for the determination ofdigoxin and related compounds in Digitalis lanata. Phytochemis-try 15:1537

Yusakul G, Kitirattrakarn W, Tanwanichkul N, Tanaka H, Putalun W(2012) Development and application of an enzyme-linked immu-nosorbent assay for specific detection of mangiferin content invarious cultivars ofMangifera indica leaves using anti-mangiferinpolyclonal antibody. J Food Sci 77:C414

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