E-ISSN: Pharmacognostic and phytochemical …...pharmacognostic evaluation as recommended by World...

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Journal of Pharmacognosy and Phytochemistry 2016; 5(5): 33-41

E-ISSN: 2278-4136 P-ISSN: 2349-8234 JPP 2016; 5(5): 33-41 Received: 07-07-2016 Accepted: 08-08-2016 Farah-Saeed Department of Pharmacognosy, Dow College of Pharmacy, Dow University of Health Sciences, Karachi-Pakistan Mansoor Ahmad Research Institute of Pharmaceutical Sciences, University of Karachi, Karachi-Pakistan Correspondence Farah-Saeed Department of Pharmacognosy, Dow College of Pharmacy, Dow University of Health Sciences, Karachi-Pakistan

Pharmacognostic and phytochemical evaluation of eight crude extracts used in complementary and

alternative system of medicine

Farah-Saeed and Mansoor Ahmad Abstract The current study focuses the pharmacognostic evaluation of Arnica montana, Cicuta virosa, Digitalis purpurea, Sambucus nigra, Thuja occidentalis, Urtica urens, Arctostaphylos uvaursi and Apis mellifera. Microscopic and histological examination, color reaction tests; thin-layer chromatography and fourier transform infra-red spectroscopy were carried out on crude extracts. Microscopic and histological examination exhibited the presence and absence of different cells or tissues (diagnostic characters). The presence of carbohydrates, proteins, steroids, saponins, tannins, alkaloids and tri-terpenes were detected in crude extracts of under studied plants and insect. In thin-layer chromatography (TLC) the presence of different chemical compounds were observed, their Rf values were calculated. Fourier transform infra-red (FT-IR) spectroscopy revealed the presence of major functional groups in each extract. Each plant material was also observed under microscope and all major diagnostic characters were recorded. Keywords: Thin-layer chromatography, fourier transform infra-red spectroscopy, morphological studies, microscopic studies, chemical constituents Introduction Drugs of natural origin have played a significant role in prevention and alleviation of different pathologies afflicting mankind since ancient times. The natural origin drugs are preferred more nowadays due to the lesser side-effects associated with its use, more structural diversity leading to the new drug molecules discovery and present validated techniques for pharmacognostic evaluation as recommended by World Health Organization. Pharmacognostic and phytochemical evaluation is mandatory for understanding the therapeutic efficacy of natural origin drugs and validating the efficacy and safety profile of the drugs [1-4]. The exploration of hidden properties of phyto-medicine is essential for emerging trends in medicine. Therefore preliminary pharmacognostic and phytochemical analysis of crude extracts of Apis mellifera (Apidae), Arctostaphylos uva-ursi (Ericaceae), Arnica montana L. (Asteraceae), Cicuta virosa (Apiaceae), Digitalis purpurea L. (Scrophulariaceae), Sambucus nigra L. (Caprifoliaceae), Thuja occidentalis L. (Cupressaceae) and Urtica urens L. (Urticaceae) were carried out. Material & Method Collection and Preparation of Crude extract The medicinal plants; Arctostaphylos uvaursi, Urtica urens, Arnica montana, Cicuta virosa, Digitalis purpurea, Sambucus nigra, Thuja occidentalis and insect drug Apis mellifera were collected from different places; they were identified by Prof. Dr. Mansoor Ahmad, Research Institute of Pharmaceutical Sciences, University of Karachi, Karachi-Pakistan and the voucher specimen (FSMP-02-09) was deposited in the herbarium of Research Institute of Pharmaceutical Sciences, University of Karachi. The plants and insect drugs were shade dried, chopped and soaked in 3L of ethanol. Alcoholic extract was prepared by percolation. This procedure was repeated thrice. Extract was concentrated by rota-evaporator (Buchi-Rotary Evaporator, Switzerland, model # B490) at 40 C. The yield of the extract was used for further experiments. Chemicals & Reagents All the chemicals and reagents used were of analytical grade and purchased from Merck (Germany).

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Journal of Pharmacognosy and Phytochemistry

Microscopic examination of crude drugs Microscopic examination of crude drugs in entire and powdered form was carried out using microscope to detect diagnostic microscopic characters like cellular tissues, calcium oxalate crystals, trichomes, stomata, starch granules. Chloral hydrate, glycerin and iodine solutions were used to differentiate cellular structures [2, 5]. Chemical Identification Tests For the detection of chemical constituents the following tests were performed, terpenes, alkaloids, tannins (lead acetate and Phenazone), saponins (froth and precipitate tests), carbohydrates (Molish test), sterols and proteins tests [6]. Thin Layer Chromatography (TLC) Thin-layer chromatography was carried out using two solvent systems: ethyl acetate-methanol-water (100: 16.5: 13.5) and chloroform-methanol-water (80: 20: 2) respectively. The chromatograms were observed under UV lamp and Rf values were calculated [7]. FT-IR spectroscopy FT-IR analysis was performed on Nicolet Avatar 330 FT-IR, USA [8]. Results The results of the phytochemical analysis of eight drug extracts are as follows: Macroscopic and microscopic evaluation Macroscopic and microscopic evaluation of an insect drug Apis mellifera and plant drugs; Arctostaphylos uvaursi, Urtica urens, Arnica montana, Cicuta virosa, Digitalis purpurea, Sambucus nigra, Thuja occidentalis were carried out and are shown in figures 1-8 respectively.

Fig 1: Apis mellifera L. and its diagnostic features: (a) Head (front) (b) Brain of A. mellifera (c) A. mellifera head showing distinct

compound eyes and antennae (d) Macroscopic view of A. mellifera (e) Microscopic over view of a whole A. mellifera section.

Fig 2: A. uva-ursi L. and its diagnostic features: (a) mesophylls near mid-rib containing calcium oxalate crystals; (b) sunken stomatas in epidermis; (c) calcium oxalate crystals; (d) flower; (e) longitudinal section of leaf; (f) fibres; (g) gynoecium; (h) twig with flower; (i)

unicellular trichomes.

Fig 3: Arnica montana L. and its diagnostic features: (a) pollen grains; (b) ray florate and disc florate; (c) pollen grains; (d) pappus

borste; (e) unicellular trichomes; (f) fruit knot on epidermis; (g) multicellular trichome (h) glandular trichomes; (i) multicellular trichomes; (j) epidermis of corolla containing oil globules; (k)

endothecium; (l) fruit peel with phytomelan

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Fig 4: Cicuta virosa L. and its diagnostic features: (a) Transverse section of C. virosa root; (b) transverse section of endosperm; (c) endosperm; (d) fruit; (e) twig with leaves, flowers and fruits; (f)

longitudinal section of C. virosa root.

Fig 5: Digitalis purpurea and its diagnostic features: (a) transverse section of midrib (b) transverse section of D. purpurea leaf (c)

glandular trichome with uniserrate stalk (d) glandular trichome with bicellular heads (e) lower epidermis (f) leaf (g) upper epidermis

Fig 6: Sambucus nigra and its diagnostic features: (a) epidermis containing sieve tubes, mesophyll cells, calcium oxalate crystals and

starch grains; (b) endothecium; (c) unicellular and grandular trichomes; (d) the vascular system; (e) sunken stomata in lower

epidermis; (f) S. nigra plant; (g) pollens.

Fig 7: Thuja occidentalis L. and its diagnostic features: (a) twig of T. occidentalis; (b) sieve tubes; (c) transverse section of leaf; (d)

sunken stomata in epidermis; (e) schizogenous cavities.

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Fig 8: Urtica urens L. and its diagnostic features: (a) transverse section of leaf containing sieve tubes and schizogenous cavities; (b) different types of trichomes; (c) trichomes with epidermis; (d) sieve

tubes; (e) schizogenous cavities; (f) sunken stomata in lower epidermis; (g) whole plant.

Fig 9: Shows the chromatogram of the eight drugs (extracts) in solvent system chloroform-methanol-water (80: 20: 2) at short wave

length 254 nm.

Fig 10: Shows the chromatogram of the eight drugs (extracts) in solvent system ethyl acetate-methanol- water (100: 16.5: 13.5) at

long wave length 366 nm.

Fig 11: Shows the FT-IR spectra of A. mellifera drug extract (significant functional groups peaks include OH, CH, benzene, C-O-C).

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Fig 12: Shows the FT-IR spectra of A. uva-ursi drug extract (significant functional groups peaks include OH, CH, benzene, C-O-C).

Fig 13: Shows the FT-IR spectra of A. montana drug extract (significant functional groups peaks include OH alcoholic, =C-H, CH, OH acidic, C=O, benzene ring, C-O-C).

Fig 14: Shows the FT-IR spectra of C. virosa drug extract (significant functional groups peaks include OH, CH, benzene, C-O-C).

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Fig 15: Shows the FT-IR spectra of D. purpurea drug extract (significant functional groups peaks include OH, CH, benzene, C-O-C).

Fig 16: Shows the FT-IR spectra of S. nigra drug extract (significant functional groups peaks include OH, CH, benzene, C-O-C).

Fig 17: Shows the FT-IR spectra of T. occidentalis drug extract (significant functional groups peaks include OH, CH, benzene, C-O-C).

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Fig 18: Shows the FT-IR spectra of U. urens drug extract. (Significant functional groups peaks include OH, CH, benzene, C-O-C).

Table 1: Chemical identification tests of the eight crude extracts using different chemical reagents.

S. No.

DRUG NAME TANNINS

(lead acetate test)

SAPONINS (froth

formation)

ALKALOIDS

CHO (Molish test)

PROTEINS STEROLS

STEROIDS

EtOH Tannic

acid Napthol -

H2SO4 H2SO4

1. Apis mellifera

Very slight ppts

Froth formation

No orange ppts

Color changes to purple Soluble No

ppts

No Purple ring

formation

Purple color

Golden brown

2. Arnica montana Very slight

ppts No froth

formation No orange

ppts Color changes

to purple Soluble No ppts

Purple ring formation

Purple color Brown

3. Cicuta virosa No ppts Froth formation

No orange ppts


ppts Purple ring formation

Purple color Golden yellow

4. Digitalis purpurea

No ppts Froth formation

No orange ppts


ppts Purple ring formation

Purple color Brown

5. Sambucus nigra

Slight fine ppts

Froth formation

No orange ppts

Color changes to purple Soluble No ppts Purple ring

formation Blackish-

green color Orange

6. Thuja occidentalis


No orange ppts


formation Purple color Brown

7. Urtica urens


No orange ppts


formation Black color Golden brown

8.

Arctostaphylos uva-ursi

Fine ppts Excessive

Froth formation

Orange ppts

Color changes to purple Soluble No ppts

No Purple ring

formation

Blackish purple color

Brown

Table 2: Rf value of the eight crude extracts

S. No. Name of drugs Rf value of drugs in different solvent systems

CHCl3: MeOH: H2O (80:20:2) Ethyl acetate: MeOH: H2O (100:16.5:13.5) At 254 nm At 365 nm At 254 nm At 365 nm

1 Arnica montana

0.03 0.04 0.01 0.03 0.07 0.11 0.11 0.11 0.17 0.19 0.15 0.22 0.26 0.29 0.23 0.33 0.30 0.42 0.30 0.39 0.36 0.54 0.35 0.45 0.47 0.67 0.39 0.57 0.74 0.76 0.56 0.67

0.80 0.60 0.74 0.66 0.74

2 Thuja occidentalis

0.03 0.01 0.01 0.03 0.05 0.05 0.07 0.08 0.11 0.14 0.12 0.30 0.16 0.26 0.23 0.39 0.28 0.40 0.26 0.56 0.33 0.56 0.32 0.61

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0.40 0.66 0.39 0.80 0.43 0.73 0.45 0.90 0.49 0.80 0.56 0.52 0.88 0.67 0.56 0.80 0.59 0.84 0.66 0.87 0.73 0.77 0.92

3 Digitalis purpurea

0.01 0.01 0.01 0.04 0.06 0.08 0.07 0.11 0.08 0.14 0.23 0.28 0.17 0.50 0.30 0.42 0.22 0.60 0.43 0.56 0.28 0.69 0.52 0.32 0.87 0.54

0.38 0.57 0.47 0.73 0.53 0.83 0.57 0.90 0.67

4 Apis mellifera

0.02 0.03 0.01 0.03 0.10 0.15

0.09 0.21

0.09 0.23

0.05 0.11

0.23 0.50 0.38 0.16 0.35 0.43 0.46 0.22 0.45 0.73 0.53 0.26 0.66 0.78 0.59 0.39

0.87 0.46 0.52

5 Urtica urens

0.02 0.01 0.05 0.03 0.11 0.08 0.16 0.09 0.14 0.16 0.28 0.23 0.22 0.25 0.35 0.38 0.28 0.36 0.42 0.45 0.35 0.52 0.49 0.53 0.46 0.70 0.57 0.59 0.50 0.66 0.91

0.71 0.76

Chemical Identification Tests Results of chemical identification tests performed on eight respective crude drugs are tabulated in table 1. Thin-layer chromatography Chromatograms and Rf values of the eight crude extracts are shown in fig.9-10 and table 2. Fourier Transform Infra-red Spectroscopy Fourier transform infra-red spectra exhibiting the functional groups of each studied crude drug are shown in figures 11-18. Discussion The crude extracts studied in our present research are being used in complementary and alternative system of medicine since long. This research work is carried out to meet the requirements of World Health Organization and other regulatory bodies concerning standardization of natural-origin drugs on same pattern as synthetic drugs. Preliminary pharmacognostic studies (macroscopy, microscopy, color reaction tests, thin layer chromatography and fourier transform infra-red spectroscopy) were carried out on the eight crude drugs respectively to authenticate the presence of chemical constituents in them reported by other researchers [9-

16].

Conclusion Our pharmacognostic and phytochemical evaluation study will facilitate in carrying out further pre-clinical and clinical trials on the selected eight drugs of natural origin. Abbreviations Used TLC – Thin-layer chromatography FT-IR – Fourier transform infra-red Rf = retardation factor HCl = hydrochloric acid H2SO4 = Sulphuric acid A. mellifera = Apis mellifera A. uva-ursi = Arctostaphylos uva-ursi A. montana = Arnica montana C. virosa = Cicuta virosa D. purpurea = Digitalis purpurea S. nigra = Sambucus nigra T. occidentalis = Thuja occidentalis U. urens = Urtica urens Conflict of Interest Authors declared no conflict of interest.

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