Int J Pharm Pharm Sci, Vol 8, Issue 1, 144-149Original Article


PHARMACOGNOSTICAL, PHYTOCHEMICAL SCREENING AND STANDARDIZATION OF NELUMBO NUCIFERA (GAERTN): THE SACRED LOTUS

ALOK BHARDWAJ1*, K. P. MODI2

1School of Pharmacy, RK University, Rajkot, Gujarat, India, 2B. K. Mody Govt. Pharmacy College, Rajkot, Gujarat, India
Email: alok.nicholas@gmail.com  

Received: 15 Sep 2015 Revised and Accepted: 18 Nov 2015

ABSTRACT

Objective: To perform the Phytochemical screening & standardization of Nelumbo nucifera Gaertn.

Methods: The present study provides pharmacognostic, physicochemical and phytochemicals details of Parts of Nelumbo nucifera. Macro & Microscopic evaluation and world health organization (WHO) recommended parameters were followed in the study.Parts of Nelumbo nucifera were used for phytochemicals evaluation & standardization. Plant parts were collected from the bank of Yamuna and hinden river from National Capital region.

Results: The Physiochemical analysis of the different plant extracts showed Extractive Values [(%w/w) ±SEM] which were compared with standard & it was found as: Alcohol soluble Extractives: 6.347±0.053, Water soluble Extractive: 20.533±0.267, Chloroform soluble Extractives: 12.533±0.533, Petroleum Ether soluble Extractives: 5.867±0.133. Other standardization parameters were found to be as: Moisture content (%w/w): 6.967±0.441, Total Ash value (%w/w) ±SEM: 5.933±0.333, Acid insoluble ash value: 0.933±0.058, Water insoluble ash value (%w/w): 2.511±0.033. The results obtained from qualitative evaluation of HPTLC fingerprint were helpful in the identification and quality control of the drug. HPTLC analysis can provide standard fingerprints and can be used as a reference for the identification and quality control of the drug.

Conclusions: The results of this study can serve as valuable source of information for identification of this plant for future investigation and applications.

Keywords: Nelumbo nucifera, Pharmacognostic, Phytochemical, Physicochemical, Standardization.

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Nelumbo nucifera Gaertn (Nelumbonaceae) known by numerous common names including Indian lotus, sacred lotus, bean of India, or simply lotus, is one of two species of aquatic plant in the family Nelumbonaceae. The Linnaean binomial Nelumbo nucifera Gaertn is the currently recognized name for this species, which has been classified under the former names, Nelumbium speciosum (Wild) & Nymphaea nelumbo, among others. Herbal medicines are represented as the most potential field of alternative medicines all over the world for a number of diseases in which allopathic medicines have no or little solution [1].

The use of herbs and herbal products, in both developing and developed countries, for the treatment of various diseases has increased dramatically in recent years. However the major drawback in promoting the use of medicinal plants is the lack of standardization as well as the confusion in the identification of the plant and their substitutes or adulterants. To ensure reproducible quality of herbal plants, authentication is invaluable. The pharmacognostical studies not only give the authentication but also quality and purity standards of the plant. According to the WHO, the macroscopical and microscopical description of a medicinal plant is the first step towards establishing the identity and degree of their purity [2].

Now-a-days there is a renewed interest in drugs of natural origin simply because they are considered as green medicine and green medicine is always supposed to be safe. Another factor which emphasizes this attention is the incidences of harmful nature of synthetic drugs which are regarded as harmful to human beings and environment. The advantage of natural drugs is their easy availability, economic and less or no side effects but the disadvantage is that they are the victims of adulteration. The more effective the natural drug more is its demand and the chances of non-availability increases. To meet the growing demand, the natural drug is easily adulterated with low grade material. Adulteration or substitution is nothing but replacement of original plant with another plant material or intentionally adding any foreign substance to increase the weight or potency of the product or to decrease its cost. Therapeutic efficacy of medicinal plants depends upon the quality and quantity of chemical constituents. The misuse of herbal medicine or natural products starts with wrong identification. The most common error is one common vernacular name is given to two or more entirely different species. All these problems can be solved by pharmacognostic studies of medicinal plants. It is very important and in fact essential to lay down pharmacognostic specifications of medicinal plants which are used in various drugs. Pharmacognosy is the study of medicines derived from natural sources, mainly from plants. It basically deals with standardization, authentication and study of natural drugs [2].

A Pharmacognostic study ensures plant identity, lays down standardization parameters which will help and prevents adulterations. Such studies will help in authentication of the plants and ensures reproducible quality of herbal products which will lead to safety and efficacy of natural products [2].

The pharmacognostic standardization parameters of Nelumbo nucifera Gaertn were performed. The parameters which are studied are moisture content, loss on drying, total ash, acid-insoluble ash, alcohol and water-soluble extractive values, petroleum ether soluble extractive value, ethyl acetate soluble extractive value, acetone soluble extractive value etc. The pharmacological studies have shown that N. nucifera possesses various pharmacological activities like anti-ischemic, antioxidant, anticancer, antiviral, anti obesity, lipolytic, hypocholestemic, antipyretic, hepatoprotective, hypoglycemic, antidiarrhoeal, antifungal, antibacterial, anti-inflammatory and diuretic activities.

Several different types of compounds have been derived from this plant belonging to different chemical groups, including alkaloids, flavonoids, glycosides triterpenoid, etc., with own therapeutic impact. Combined gas/liquid chromatography–mass spectroscopy has shown that the parts of the plant are rich in a number of alkaloids. In the analysis of non-phenolic fractions of the leaf extract, the major components had retention data and mass spectra identical to those of nuciferine, roemerine, anonaine, pronuciferine and N-nornuciferine [3].

Dehydroemerine, dehydronuciferine, dehydroanonaine, pronuciferine, N-nornuciferine, O-nornuciferine, nuciferine, remerine, armepavine, liensinine, isoliensinine, negferine, asimilobine and lirinidine were isolated from leaves and petioles [4-7]. The leaves also contain a glycoside, nelumboside, and flavonoids such as quercetin and leuco-anthocyanidin which were identified as leucocyanidin and leucodelphinidin [8, 9]. The presence of some other flavonoids in the leaves such as quercetin 3-O-a-arabinopyranosyl-(1,2)-galacto-pyranoside, quercetin-3-O-D-glucuronide, rutin,(+)-catechin, hyperoside, isoquercitrin and astragalin has also been reported [4].

MATERIALS AND METHODS

Plant material

Plant was identified and collected. Dust and debris were removed from the plant parts and dried in air for complete drying. Parts of Nelumbo nucifera were used for pharmacognostic evaluation. Plant parts were collected from the bank of Yamuna and hinden river from NCR. Plant was identified and authenticated by the Dr Anjula Pandey principal scientist National Herbarium of Cultivated Plants, Division of plant exploration & germ plasm collection, National Bureau of Plant Genetic Resources, Pusa Institute, New Delhi.

Preparation of extract

The plant parts were dried and ground. Powder of dried part was made by a domestic grinder. Powder is used for various extractions.

  1. Petroleum Ether extract
  2. Chloroform extract
  3. Methanol extract
  4. Aqueous extract

The powdered drug of above mentioned plant has been subjected to successive solvent extraction, with petroleum ether, chloroform, methanol and water. After complete extraction, the solvent was evaporated under vacuum at room temperature to yield semisolid. The percentage yield, color, and consistency were determined. The extract was then preserved in a desiccator till further use.

Preliminary test like test for alkaloids, flavonoids, saponins, steroids etc were performed. After the Preliminary test TLC of the extracts has been carried out followed by HPTLC Fingerprint Profile of Alcoholic Extract of the plant. Thin layer chromatographic (TLC) studies were performed using various solvent systems with different ratio were tried. The solvent system which showed the best separation of the components was selected for the extract. The TLC procedure was optimized with a view to evaluate the herbal extract.

RESULTS

The various standardization parameters of the plant were determined viz organolepticcharacters,macro & microscopic studies, Physico-chemical analysis etc.

Organoleptic characters

Organoleptic evaluation can be done by means of sense organs, which provide the simplest as well as quickest means to establish the identity and purity to ensure quality of a particular drug. Organoleptic characters such as shape, size, colour, odour, taste and fracture of stem bark, leaf structure like margin, apex, base surface, venation and inflorescence, etc are evaluated.

Macroscopic study

The macroscopic study is the morphological description of the plant parts which are seen by naked eye or magnifying lens. Nelumbo nucifera Gaertn a large, aquatic herb with creeping stem, occurring throughout warmer parts of the country up to an altitude of 1000 m. Drug occurs as entire or pieces of flowers, comprising of calyx, corolla, androecium, gynoecium and thalamus; entire flower 10-15 cm in dia., yellowish-brown; sepals leaf-like, crimpled, 3-5 cm long, 1.3-2 cm wide, dark brown, broken pieces also occur; petals numerous, crimp led, elliptic, obtuse, membranous, finely veined, 2-4 cm long, 1.2-2 cm wide yellowish-brown; anther, erect, linear 1.4-2 cm long, extended into clavate appendages; gynoceium apocarpous; carpels many, free, embedded in a creamy, top shaped fleshy thalamus (torus) 3-5 cm long and 2.5-3 cm wide; fruit anetaerio of achenes, becoming loose in their sockets when ripe; seed hard, black, starchy and large [10].

Microscopic study

The microscopic study is the anatomical study which is done by taking appropriate section of the plant parts under study. Each distinguishing character can be noted down, some of which are retained in the powder study also. Some of the chemicals which are used in obtaining clear sections are phloroglucinol, chloral hydrate, safranine, methyl orange, etc.

Flower

Petal-shows single layered epidermis on both surfaces, consisting of rectangular cells covered with striated cuticle; ground tissue consisting of polygonal, parenchymatous cells with wide air-sacs.

Stamen

Filament-filament appears circular in outline, consisting of single layered epidermis covered with striated cuticle; followed by ground tissue of oval, angular, parenchymatous cell; vascular bundle single, present in centre consisting of usual elements of xylem and phloem tissues.

Anther

Shows four chambered anther, two on either sides, connected by parenchymatous cells containing vascular bundle; anther consists of a single layer of epidermis, composed of thin-walled, rectangular, parenchymatous cells followed by single layer of endothecium consisting of thin-walled, columnar, parenchymatous cells; spore sac contains yellow, spherical pollen grains with smooth exine and intine walls, measuring 50-61 μ in diameter.

Powder

Dusty brown; shows fragments of vessels with spiral thickening, spherical, yellow pollen grains, measuring 50-61 μ in dia. having smooth exine and intine.

Table 1: Identity, purity and strength

Foreign matter

Not more than 2 per cent (Ayurvedic pharmacopoeia Appendix 2.2.2.)

Total ash

Not more than 12 per cent (Ayurvedic pharmacopoeia Appendix 2.2.3.)

Acid-insoluble ash

Not more than 3 per cent (Ayurvedic pharmacopoeia Appendix 2.2.4.)

Alcohol-soluble extractive

Not less than 6 per cent (Ayurvedic pharmacopoeia Appendix 2.2.5.)

Water-soluble extractive

Not less than 14 per cent (Ayurvedic pharmacopoeia Appendix 2.2.6.)

Powder study

Powder study is similar to microscopic study except here dried powder is taken instead of section of the plant. All the reagents used are also same like above.

Physico-chemical analysis

Ash values are used to determine quality and purity of crude drug. It indicates presence of various impurities like carbonate, oxalate and silicate. The water soluble ash is used to estimate the amount of inorganic compound present in drugs. The acid insoluble ash consist mainly silica and indicate contamination with earthy material. Moisture content of drugs should be at minimal level to discourage the growth of bacteria, yeast or fungi during storage. Estimation of extractive values determines the amount of the active constituents in a given amount of plant material when extracted with a particular solvent. The extractions of any crude drug with a particular solvent yield a solution containing different Phyto-constituents. The compositions of these Phyto-constituents depend upon the nature of the drug and the solvent used. It also gives an indication whether the crude drug is exhausted or not.

3

A: Parenchymatous cells of Nelumbo nucifera Gaertn

4

B: Lignified fibre of  Nelumbo nucifera Gaertn

5

C: Epidermis of Nelumbo nucifera Gaertn

7

D: Xylem vessel of Nelumbo nucifera Gaertn

Fig. 1: Powder microscopy of Nelumbo nucifera Gaertn


Table 2: Extractive values of Nelumbo nucifera Gaertn

Sample Nelumbo nucifera Gaertn

Extractive value (%w/w)±SEM

Reference value (As per IP 2007& ayurvedic pharmacopoeia)

Alcohol soluble Extractives

6.347±0.053

Alcohol-soluble extractive Not less than 6 %

Water soluble Extractives

20.533±0.267

Water-soluble extractive Not less than 14 per cent

Chloroform soluble Extractives

12.533±0.533

____

Petroleum Ether soluble Extractives

5.867±0.133

_____

Values are mean of three independent replicates±Standard deviations


Table 3: Ash values of Nelumbo nucifera Gaertn

(Sample) Nelumbo nucifera gaertn

Ash value (%w/w)±sem

Reference value (as per IP 2007&ayurvedicpharmacopoeia)

Moisture content

6.967±0.441

Not more than 15%

 

Ash values

5.933±0.333

Total Ash Not more than 12 per cent
 

Acid insoluble ash value

0.933±0.058

Acid-insoluble ash not more than 3 %
 

Water insoluble ash value

2.511±0.033

____

Values are mean of three independent replicates±Standard deviations

Phytochemical analysis

The crude powder and/or crude drugs extracted in different solvents are tested for various Phyto-constituents present in them by standard procedures. They are generally tested for the presence of alkaloids, flavonoids, tannins, phenols, cardiac glycosides, triterpenes, steroids and saponins. The powder of dried rhizome was subjected to continuous Soxhlet’s extraction with various organic solvents such as petroleum ether (60-80C), chloroform, benzene, methanol & ethanol respectively.

After concentration and drying of each extract in vacuum desiccator identification of Phyto-constituents was carried out using thin layer chromatography method by different detecting reagents [3].

Table 4: Phytochemical screening of different extracts Nelumbo nucifera gaertn

Experiment

Test

Aqueous Extract

Alcoholic extract

Chloroform extract

Pet. Ether Extract

Inference

Test for Carbohydrates

Molisch’s test

+

+

+

-

Carbohydrate

Fehling’s test

+

+

+

-

Reducing sugar

Benedict’s test

+

-

-

-

Reducing sugar

Barford’s test

+

-

-

-

Monosaccharide

Cobalt-chloride Test

+

-

-

-

Glucose

Tollen’s phloroglucinol test

+

-

-

-

Hexose sugar

Test for Starch

(Cold Extract)

(Hot Extract)

+

+

-

-

-

Starch

Test for Proteins and Amino Acid

Ninhydrin test

+

-

-

-

Protein or amino acid

Biuret test

+

-

-

-

Polypeptide

Test with Tannic Acid

+

-

-

-

Protein

Xanthoprotein test

+

-

-

-

Protein

Test for Fixed oils

Spot Test: (a)Benzene extract

-

-

-

+

Fixed oil

(b)Petroleum ether extract

-

+

-

+

Fixed oil

Test for Phytosterols

Liebermann’s test

-

+

-

+

Phytosterol

Liebermann-burchard’s test

-

-

-

+

Phytosterol

Salkowski’s test

-

+

-

-

Phytosterol

Test for Glycosides

Baljet’s test

+

+

-

-

Glycoside

Legal’s test

+

-

-

-

Glycoside

Borntrager’s test

-

-

-

-

Anthraquinone glycoside

Modified borntrager’s test

-

-

-

-

Anthraquinone glycoside

Kedde’s test

-

-

-

-

Cardenolide

Killer kiliani test

-

-

-

-

α-deoxy sugar

Test for Saponins

Foam test

+

-

-

-

Saponins

Test for Flavonoids

Ferric chloride test

-

-

-

-

Flavonoids
 

Lead acetate test

-

-

-

-

Flavonoids
 

Action of Alkali and Acid

-

+

-

-

Flavonoids

Test for Tannins & Phenolic Compounds

 Ferric chloride test

+

-

-

-

Phenolic compound
 

Nitric acid test

+

+

-

-

Tannins and Phenolic compound present
 

Gelatin test

+

+

-

-

Tannins
 

Match stick test

+

-

-

-

Tannins
 

Chlorogenic acid Test

-

+

+

-

Tannins

Test for Alkaloids

Mayer’s test

+

+

+

-

Alkaloids
 

Dragendorff’s test

+

+

+

-

Alkaloids
 

Wagner’s test

+

+

+

-

Alkaloids
 

Hager’s test

+

+

+

-

Alkaloids

+= Present; -= Absent

Development of the optimum mobile phase by TLC

HPTLC fingerprint for estimation of quercetin in Nelumbo nucifera extract

The solvents were purified prior to chromatographic analysis for a good separation of Phyto-constituents.

The alcoholic extract of N. nucifera was chromatographed on silica gel 60 F254 plates with toluene: ethyl acetate: formic acid 5: 4: 0.2 (v/v/v), as mobile phase. The peak purity of quercetin was assessed by comparing the spectra at peak start, peak apex and peak end positions of the spot. Good correlation was obtained between the standard and the sample. The identity of the quercetin bands in sample chromatograms was confirmed by the chromatogram obtained from the sample with that obtained from the reference standard solution (Figure) and by comparing retention factors of quercetin from sample and standard solutions. The peak corresponding to quercetin from the sample solution (Rf 0.65) had same retention factor as that from the quercetin standard (Rf 0.68) (fig. 3). The proposed HPTLC method provides a good resolution of quercetin from other constituents present in alcoholic extract of dried flowers of N. nucifera.


1

Fig. 2: TLC of Nelumbo nucifera

2

Fig. 3: HPTLC of Nelumbo nucifera

3

Fig. 4: HPTLC Fingerprinting for estimation of quercetin in Nelumbo nucifera

DISCUSSION

Natural products are both a fundamental source of new chemical diversity and an integral part of today’s pharmaceutical compendium. About 75-80 % of the world’s population in developing countries mainly depends on herbal medicine for primary health care because of better compatibility with the human body and less side effects [15].

For the purposes of quality control, assessment of purity and identification of any sample, standardization is very much essential. In the present research, pharmacognostic study, physiochemical analysis of Nelumbo nucifera plant parts was carried out. Pharmacognostical studies and determination of different physiochemical parameters are very much essential for the standardization of drug and establishing its pharmacological efficacy. Hence, these studies help in identification and authentication of the plant material [16-17]. Phytochemicals analysis of Nelumbo nucifera plant revealed the presence of carbohydrate, glycoside, proteins and amino acids, flavonoids, tannins & phenol compounds, alkaloids, fixed oils, and phytosterols. The literature showed that Nelumbo nucifera plant is rich inwide variety of metabolites such as phenols & other compounds.

The results obtained from qualitative evaluation of HPTLC fingerprint were helpful in the identification and quality control of the drug. HPTLC analysis can provide standard fingerprints and can be used as a reference for the identification and quality control of the drug.

This research article described many Pharmacognostical features of Nelumbo nucifera Gaertn which will give a new direction for the establishment of novel compounds which would be beneficial for scientific research. Nelumbo nucifera Gaertn is an important drug of indigenous system of medicine and has been known for a number of medicinal properties in ayurveda but still its pharmacological potential has not been fully explored. This research article tried to provide scientific basis for the further development of Nelumbo nucifera Gaertn.

CONCLUSION

The results of this study can serve as valuable source of information for identification of this plant for future investigation and applications. Thus we conclude that this approach is a new innovative idea to design and screen the Nelumbo nucifera Gaertn which has given a new direction for the establishment of newer compounds which would be beneficial for the mankind.

ACKNOWLEDGEMENT

The first author Alok Bhardwaj is thankful to the Dr. K. P. Modi (Associate Professor) at B. K. Mody Govt. Pharmacy College, Rajkot, India, for providing general support and encouraging my work. We also thank Taxonomists for identifying the plant species used in the study.

CONFLICT OF INTERESTS

We declare that we have no conflict of interest.

REFERENCES

  1. Shen MJ, Schopf JW, Harbottle G, Cao RJ, Ouyang S, Zhou KS, et al. Long-living lotus: Germination and soil irradiation of centuries-old fruits, and cultivation, growth, and phenotypic abnormalities of offspring. Am J Bot 2002;89:236–47.
  2. Chanda S. Importance of pharmacognostic study of medicinal plants: An overview. J Pharmacogn Phytochem 2014;2:69-73.
  3. Kunitomo J, Yoshikawa Y, Tanaka S, Imori Y, Isoi Y, Masada Y, et al. Alkaloids of Nelumbo nucifera. Phytochemicals 1973;12:699–701.
  4. Kashiwada Y, Aoshima A, Ikeshiro Y, Chen YP, Furukawa H, Itoigawa M, et al. Anti-HIV benzylisoquinoline alkaloids and flavonoids from the leaves of Nelumbo nucifera, and structureactivity correlations with related alkaloids. Bioorg Med Chem 2005;13:443–8.
  5. Xubiao Luo, Bo Chen, Jingjing Liu, Shouzhuo Yao. Simultaneous analysis of N-nornuciferine, O-nornuciferine, nuciferine and roemerine in leaves of Nelumbo nucifera Gaertn by high-performance liquid chromatography: photodiode array detection-electrospray mass spectrometry. Anal Chim Acta 2005;538:129–33.
  6. Tomita M, Furukawa H, Yang TH, Lin TJ. Studies on the alkaloids of lotus embryo & alkaloids of Nelumbo nucifera Gaertn. Chem Pharm Bull 1965;13:39-43.
  7. Shoji N. Asimilobine and liridine, serotonergic receptor antagonists from Nelumbo nucifera. Nat Prod 1987;50:773-4.
  8. Nakaoki T. Medicinal resources XIX: Flavonoid of the leaves of Nelumbo nucifera, Cosmos hipinnatus and Foeniculum vulgare. Yaku Zas 1961;81:1158-9.
  9. Nagarajan S. Chemical examination of the flowers of Nelumbium speciosum wild. Curr Sci 1966;35:176.
  10. He LS, Meng FL, Diao XJ, Li YW, Meng R, Xi BD, et al. Allelopathic effects of Nelumbo nucifera stem and leaf tissue extract on the growth of microcystis aeruginosa and Scenedesmus quadricanda. Huanjing Kexue 2013;34:2637-41.
  11. Department of Indian System of Medicine and Homoeopathy New Delhi. Ayurvedic Pharmacopoeia of India; 2001;1:143-7.
  12. Kyari MZ. Extraction and characterization of seed oils. Int Agrophys 2008;22:139-42.
  13. Evans WC. Trease and evans pharmacognosy 16th ed. London: Saunders Elsevier; 2009.
  14. Harborne JB. Chapmann and Hall London. Phytochemical Methods. 2nd ed. A Guide to Modern Techniques of Plant Analysis; 1984. p. 192.
  15. Shinde V, Dhalwal K, Mahadik KR. Some issues related to pharmacognosy. Pharmacogn Rev 2008;3:1-5.
  16. Kumar S, Kumar V, Prakash OM. Pharmacognostic study and anti-inflammatory activity of Callistemon lanceolatus leaf. Asian Pac J Trop Biomed 2011;1:177–81.
  17. Thomas S, Patil AG, Chandra N. Pharmacognostic evaluation and physicochemical analysis of Averrhoa carambola L. Fruit. J Herb Med Toxicol 2008;2:51–4.