Lvi, 5 contributions to the pharmacognostical study on grapes Hamburg cultivar




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FARMACIA, 2008, Vol.LVI, 5



CONTRIBUTIONS TO THE PHARMACOGNOSTICAL STUDY ON Grapes Hamburg cultivar
RUAA AZIZ 1*, MARIA-LIDIA POPESCU2, DENISA MIHELE1

University of Medicine and Pharmacy "Carol Davila", Faculty of Pharmacy, 6 Traian Vuia street, Bucharest

1Department of clinical laboratory and food safety

2Pharmacognosy. Phytochemistry. Phytotherapy Department

* angel_nosi@yahoo.com

Abstract

The purpose of this paper is to report on the comparative pharmacognostical research on epicarp, mesocarp, and seeds of grapes Hamburg cultivar.

Microscopically we identified the following specific histologic elements: cells with moniliform walls, oil and epicarp with a smooth cuticle. The chemical analysis has shown the presence of procyanidins, anthocyanins, flavonoids, hydroxycinnamic acid derivatives, triterpenes, sterols, tannins, polysaccharides, monosaccharides and non-alkaloid nitrogen containing compounds. The grapes of Hamburg cultivar have a higher percent of total hydroxycinnamic acid derivatives in seeds, and a higher percent of procyanidins in epicarp.

Rezumat

Această lucrare are în vedere studiul farmacognostic comparativ al epicarpului, mezocarpului şi seminţelor strugurilor din soiul Hamburg.

Microscopic au fost identificate următoarele elemente histologice specifice: celule cu pereţi îngroşaţi moniliform, picături de ulei şi epicarp cu cuticulă netedă. Studiul chimic a evidenţiat prezenţa proantocianilor, antocianozidelor, flavonelor, acizilor fenolcarboxilici, triterpenelor, sterolilor, taninului, ozelor, poliholozidelor şi compuşilor azotaţi nealcaloidici. Strugurii din soiul Hamburg au un conţinut ridicat de derivaţi ai acizilor hidrocinamici în seminţe şi de proantociani în epicarp.



Introduction

The medicinal parts from Vitis vinifera L., grape, Vitaceae, are the leaves, the fruit and the juice. Grape seed extracts have shown the following effects: antiatherosclerotic, antitumor, antioxidant, collagen stabilization, cytoprotective, hair growth, hepatoprotective, anti-ischemic. Grape preparations are used in venous diseases and blood circulation disorders [1, 2, 6, 7, 8].

The purpose of this paper is to report the comparative pharmacognostical research on fruits (epicarp, mesocarp, and seeds) from cultivar Hamburg.

MaterialS and methods

The plant material used for the comparative pharmacognostic study was harversted from Romania in September 2007.

The identity of the product was established using:


  1. the macroscopic examination (to verify the morphological characteristics);

  2. the microscopic examination of the powder (clarified with chloralhydrate 80%). Microphotographs were taken using a photocamera (Carl-Zeiss, oc.10X, ob.40X);

3. the qualitative analysis; the products were successively extracted with the following solvents: ethyl ether, methanol and water. The extracts were used for the identification of the most important active principles. For this examination we used specific chemical reactions according to the literature. We used thin layer chromatography (TLC) on silica gel GF254 (Merck) for the identification of phenols [3, 4].

Parametrics of TLC

  • For phenols

  • mobile phase: ethyl acetat / water / formic acid / acetic acid (72/14/7/7);

  • reference solution (0.1% in methanol): rutin, hyperoside, quercitrin, caffeic acid, cholorogenic acid, rosmarinic acid;

  • Detection: spraying with methanolic solution 0.1% of diphenylboriloxyethylamine and methanolic solution 0.1% of propylene glycol and UV light (366 nm).

  • For triterpenes

  • mobile phase: chloroform/ acetone (8/2);

  • reference solution(0.1% in methanol): oleanolic acid, ursolic acid;

  • Detection: spraying with acetic anhydride and sulphuric acid/ethanol (1:1).

In order to study the compounds from the methanolic solution, the mixture of herbal product and methanol were heated (10 min.) under a reflux condenser.

The quality of the herbal drug was estimated using the following tests:

- loss on drying (according to European Pharmacopoeia 5-th edition and Romanian Pharmacopoeia Xth Edition, the method: drying in an oven at 105C);

- soluble substances (in alcohol and water, according to Romanian Pharmacopoeia Xth Edition);

- the assay for flavonoids (using a spectrophotometric method based on the chelating reaction with aluminium chloride, according to Romanian Pharmacopoeia Xth Edition, Cynarae folium monograph, based on a standard callibration curve obtained using rutin), hydroxycinnamic acid derivatives (using a spectrophotometric method based on the formation of oxymes in the presence of the sodium nitrite and sodium molybdate, according to European Pharmacopoeia 5th edition – Ash leaf monograph), procyanidins (according to European Pharmacopoeia 5th edition – Hawthorn berries monograph), and anthocyanins (according to European Pharmacopoeia 5th Edition – Bilberry fresh fruit, monograph). The results of the quantitative chemical researches were calculated on a dry basis [9, 10]. For the spectrophotometric assay a UV-VIS Cecil Series 2000 spectrophotometer was used. Statistical analysis was performed using Anova test at a significance level of 0.05 (Microsoft Excel 2003).
Results and Discussion

The macroscopic examination confirmed the identity of the raw material [7]. The analysed product consists of the fruit oblong, dark-blue-violet with seeds pear-shaped, with hard skin and two long dimples on the side.

At the microscopic examination we observed: cells with moniliform walls, oil and epicarp with a smooth cuticle (fig. 1-4)

Anatomic tissues from grapes Hamburg cultivar:







Figure 1

Parenchyma with oily drops, cells with monoliform thickened walls



Figure 2

Epicarp -detail













Figure 3

Parenchyma with vegetal pigments and oily drops



Figure 4

Epicarpal fragment with large cells, with sinuous and strongly cutinized walls


The chemical qualitative analysis has shown the presence in grapes from Hamburg cultivar of procyanidins, anthocyanins, flavonoids, hydroxycinnamic acid derivatives, polysaccharides, triterpenes, sterols, tannins, polysaccharides and non-alkaloid nitrogen containing compounds (table I). The scientific data mention these compounds in grapes [1, 5, 8]. Coumarines, carotenoids, 1,8 dihydroxyanthraquinone derivates and alkaloids were not identified.


Table I

The results of qualitative chemical analysis

Active principles
Hamburg cultivar

epicarp

mesocarp

seeds

alkaloids

-

-

-

anthocyanins

+

-

-

1,8-dihydroxyanthraquinone derivatives

-

-

-

catechic tannins

+

+

+

carotenoids

+

-

-

coumarins

+

+

+

flavonoids

+

+

+

hydroxycinnamic acid derivatives

+

+

+

non-alkaloids nitrogen-containing compounds

+

+

+

polysaccharides

+

+

+

procyanidins

+

+

+

reducing-compounds

+

+

+

sterols

+

+

+

tannins

+

+

+

triterpenoid saponins

+

+

+

Legend: + positive reaction; - negative reaction

The TLC analysis of the sterols and triterpenes made possible the separation of oleanolic acid (Rf = 0.75, violet colour and yellow fluorescence) for semen, epicarp, mesocarp. This compound was not mentioned in the available scientific literature regarding grapes [1, 5, 8]. The other triterpenes (Rf = 0.78 and Rf = 0.89) were identified; these compounds remained unidentified because we did not have the appropriate reference substances.

The study of polyphenols by TLC revelead the presence, in this cultivar, of:


  • two flavonoid compounds (Rf = 0.48, and Rf = 0.67, yellow colour and yellow fluorescence) from epicarp;

  • one flavonoid compound (Rf = 0.48) from mesocarp;

  • chlorogenic acid (Rf=0.38) in semen, epicarp, and mesocarp (this molecule was not mentioned in the available scientific literature about Vitis vinifera);

  • other four phenolcarboxilic acids (Rf = 0.25, Rf = 0.71, Rf = 0.82, Rf = 0.85; blue colour and blue fluorescence) from semen.

The results of the quantitative chemical analysis, calculated on a dry basis, are shown in table II.

Table II

Results (g%) of the comparative quantitative chemical analysis



Parameter

Hamburg cultivar

epicarp

mesocarp

seeds

procyanidins g%

4.6766+0.2635

0.3566+0.0241

0.9833+0.0088

anthocyanosides g%

0,6959+0.0712

-

-

hydroxycinnamic acid derivatives (expressed as chlorogenic acid) g%

1.6513+0.1514

2.057+0.0943

6.3166+0.1033

flavonoids

(expressed as rutin) g%



0.0281+0.0068

0.0464+0.0025

0.0196+0.0022

This study showed that the seeds of Hamburg cultivar have a higher percentaje of total hydroxycinnamic acid derivatives (6.3166+0.1033%) compared to the mesocarp (1.6513+0.151 g%) and the epicarp (2.057+0.094 g%). The epicarp of this cultivar has a higher percent of procyanidins (4.6766+0.2635 g%) compared to the epicarp (3.63 + 0.287 g%) and the seeds (1.84 + 0.314 g%). The differences regarding the content in total hydroxycinnamic acid derivatives and the content in proanthocyanidins, were not statistically significant (p > 0.05).

For flavonoids there were established a statistically significant difference between the analyzed products (p < 0.02).

CONCLUSIONS

The grapes of Hamburg cultivar have a higher percentaje of total hydroxycinnamic acid derivatives in seeds, and a higher percentaje of procyanidins in epicarp.


REFERENCES

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