Abstract
Objective
To establish the standardization parameters for complete pharmacognostic evaluation of stems of Thespesia lampas (T. lampas) (Cav.) Dalz & Gibs (Malvaceae), an important plant in the Indian system of medicine.
Methods
Morphological, microscopical, physico-chemical evaluations, florescence analysis of T. lampas stems were investigated and preliminary phytochemical analysis, GC-MS analysis and HPTLC fingerprinting were carried out for qualitative phytochemical evaluation of various extracts of stems of T. lampas.
Results
Chemo-microscopy revealed the presence of lignin, starch grains and calcium oxalate crystals. Physico-chemical evaluation used to determine numerical standards showed a result with total ash (9.03 ± 0.05) % w/w, acid insoluble ash (1.50 ± 0.01) % w/w, water soluble ash (2.51 ± 0.02) % w/w, sulphated ash (7.50 ± 0.01) % w/w, ethanol soluble extractive (0.24 ± 0.02) % w/w, water soluble extractive (0.08 ± 0.01) % w/w, moisture content (6.03 ± 0.05) % w/w and total crude fibre content of stem powder (47.36 ± 0.32) % w/w. Behavior characteristics of the stem powder showed presence of steroids, starch, alkaloid, flavonoids and proteins. Preliminary phytochemical analysis revealed presence of glycosides, phenolic compounds, tannins, steroids, saponins, flavonoids, carbohydrates and proteins. GC-MS analysis showed the presence of fatty acids such as dodecanoic acid, tetradecanoic acid, n-hexadecanoic acid, 9-tetradecenal and HPTLC fingerprinting revealed the presence of β-sitosterol and quercetin in stems of T. lampas.
Conclusions
The pharmacognostic standardization of T. lampas is useful towards establishing standards for quality, purity and sample identification.
Keywords: Thespesia lampas, Stems, Pharmacognosy, Malvaceae, Physicochemical analysis, Preliminary phytochemical testing, GC-MS analysis, Phelloderm, Periderm, Xylem
1. Introduction
Thespesia lampas (T. lampas) (Cav.) Dalz & Gibs, synonym: Hibiscus lampas Linn, is a medicinally important plant of the Malvaceae family commonly known as ‘Ranbhendi’. It has been found throughout India and in Eastern Tropical Africa[1]. It is an annual undershrub having reddish brown colored bark. Leaves are 7.5–15.0 cm long, cordate or truncate at the base; 3-lobed and lobes triangular, acuminate, finely reticulately veined sometimes with black grandular dots on the lower surface, subglabrous on the upper. Flower is yellow with crimson centre, 7–10 cm, diameter, 1–3 together at the end of long, auxillary or terminal peduncle. Fruits capsules are dull black, 2.5 cm long, ovoid, pointed, woody 4–5 valved, pilose. Seeds are glossy, glabrous, many are dark brown or black, club shaped 3 mm long[2],[3]. In the folk medicine, this plant has been considered to be hepatoprotective and traditionally root paste was used to cure jaundice in Korku tribe of Amravati district of Maharashtra and also in Nepal[4],[5]. The roots of this plant are reported for anti-diabetic, anti-hyperlipidaemic, hepatoprotective, antioxidant and anthelmintic activity[6]–[11]. The stem parts are used in the treatment of inflammation, acidity, bleeding nose, bronchitis, cough, dysentery, fever, sun stroke, urinary complaints, anthelmintic and carbuncle[12]. Stems of T. lampas have been reported for presence of gossypol[13] and its antimicrobial activity[14]. The literature survey and screening of scientific data revealed that although T. lampas stems are traditionally used in the treatment of various diseases for a long time, no systematic pharmacognostic and physico-chemical standardization has been done. The present investigation of T. lampas is therefore taken up to establish certain botanical and chemical standards like pharmacognostic characterization, physicochemical analysis and preliminary phytochemical testing of stems which would helps to prepare a monograph for the proper identification of the plant.
2. Materials and methods
2.1. Plant material
The plant, T. lampas was collected in Trimbakeshwar Hills, Nashik District (Maharashtra) in May 2008. The plant was authenticated and herbarium was deposited in Botanical Survey of India, Pune, Maharashtra under voucher specimen number CDSTL1. (Ref. No. BSI/WC/Tech/2008/79). The stems of the plant were dried, powdered and passed through 40 mesh sieve and stored in an airtight container for further use.
2.2. Macroscopic and microscopic analysis
Macroscopic studies were done by using simple microscope. The color, odour, taste, size and shape of stems were determined. Microscopic studies were done by preparing thin hand section of stems of T. lampas. The sections were cleared with chloral hydrate solution, stained with phloroglucinol- hydrochloric acid (1:1) and toludine blue[15],[16]. Powder (#60) of the dried stems was used for the observation of powder microscopical characters. The powdered drug was separately treated with phloroglucinol-hydrochloric acid (1:1) solution, acetic acid and iodine solution to determine the presence of lignified fibres, calcium oxalate crystals and starch grains respectively[15].
2.3. Physico-chemical analysis
Physico-chemical parameters of the powdered drug such as total ash, water-soluble ash, acid-insoluble ash and sulphated ash were determined. Alcohol and water-soluble extractive values were determined to find out the amount of water and alcohol soluble components. The moisture content was detected by loss on drying method[17]. Crude fibre content of stem powder was also determined[18].
2.4. Florescence analysis
Powdered stem material was analyzed under visible light, short ultra-violet light, long ultra-violet light after treatment with various organic/inorganic reagents like NaOH, HCl, HNO3 and H2SO4[19],[20].
2.5. Behaviors of stem powder
Behaviors of stem powder of T. lampas with different chemical reagent were performed to detect the occurrence of phytoconstituents along with color changes under ordinary daylight by standard method[21].
2.6. Quantitative determination of heavy metal and minerals
A total of 500 mg of air-dried stem powder was taken to determine major heavy metal and minerals content. Stem ash was also prepared by taking 2 g of sample and keeping in muffle furnace at 150 degree centigrade till constant weight was obtained. The major inorganic constituents of stem powder and ash were determined quantitatively by atomic absorption spectrometer (ASS) (Perkin Elmer-400), using argon as the carrier gas and flow rate was kept as 1 mL/2 min[22], [23].
2.7. Preparation of extract and preliminary phytochemical analysis
The air-dried stems of T. lampas were made into coarse powder. The powdered material was defatted with petroleum ether. The defatted material was extracted with methanol and distilled water using a Soxhlet extractor. Methanolic extract was further fractionated with ethyl acetate to get ethyl acetate soluble and ethyl acetate insoluble fractions. Then the extract was filtered through muslin and the filtrate was evaporated under reduced pressure and vacuum-dried[24]. The qualitative chemical test of various extracts of T. lampas was carried out using standard procedure[25].
2.8. GC-MS analysis of saponifiable matter of petroleum ether extract
The petroleum ether extract obtained was processed for separation of the saponifiable and unsaponifiable matter. 1.5 g of vacuum-dried extract was allowed to saponify using alcoholic KOH with reflux and then it was extracted with solvent ether for separation of unsaponifiable matter. The aqueous phase was acidified with concentrated H2SO4 and then again extracted with the solvent ether for separation of the saponifiable matter. All the three part were separated, i.e. petroleum ether, unsaponifiable matter and saponifiable part. The saponifiable matter of the petroleum ether extract was subjected to gas chromatography – mass spectroscopy (GC-MS)[26].
2.9. HPTLC fingerprinting of different extracts of T. lampas stems
20 mg/mL solution of petroleum ether extract, methanolic extract, ethyl acetate soluble fraction of methanolic extract and aqueous extract was prepared in petroleum ether, methanol, ethyl acetate and water respectively. Dissolve 10 µg of standard drug, β-sitosterol and quercetin in 1 mL methanol. Take 1 mL of above solution and diluted to 10 mL with methanol (0.1µg/mL). Then 20 µL was applied on the silica gel GF254 HPTLC plates (10×10). For β-sitosterol, toluene: methanol: ethyl acetate (9:1:0.5) and for quercetin, toluene: ethyl acetate: formic acid (9:1:0.5) was used as mobile phase. After development the plates were scanned in ultraviolet range at 254 nm and 366 nm and then the plates were derivatized by using 10% ethanolic sulphuric acids[27].
3. Results
3.1. Macroscopic characteristics
The stems of T. lampas have reddish brown colour. Outer bark of stem consisting of reddish brown colour with yellowish white wood. Stems have characteristic odour, bitter in taste and straight, erect, stout, smooth textured with varying length from 3–6 cm and 0.5–1.5 cm in thickness.
3.2. Microscopic characteristics
The plant, T. lampas showed the general characteristics of a dicot plant. The microscopical study of stem showed very large pith and vascular bundles arranged in a ring. The stem has thick hallow, continuous cylinder of secondary xylem and secondary phloem. A narrow periderm is evident. The lenticels present in a periderm which are similar in function to stomata, having open pores with absence of guard cells. The upper epidermal cork consists of thick walled tangentially elongated cells. Below that the presence of thin walled parenchyma with very small intracellular spaces of cortex contains bundle of acicular calcium oxalate crystals. The phloem consists of isodiametric parenchyma with small intercellular spaces. Multiseriate, thin walled, elongated medullary rays with presence of acicular and cluster form of starch grains. Pith consists of thin walled parenchymatous mucilage cavities (Figure 1a). The stem bark of T. lampas consists of two distinct regions. They are outer bark and the inner bark. Outer bark constitutes the periderm and the inner bark comprises of the secondary phloem region. Periderm consists of the outer 4 or 5 thick walled, suberised phellem cells and inner 2 to 3 layers of thin walled phelloderm cells. The periderm is crushed and appears as a dark thick band. Inner to the periderm is the secondary phloem region which consists of cone shaped dense solid bands of fibers extending up to the periderm region. The phloem consists of thick walled sclereid masses alternating with narrow band of thin walled cells of sieve elements. Phloem rays are wide and alternate with in phloem sclerenchyma cones (Figure 1b). The secondary xylem consists of vessels, xylem fibers and thin radial lines of xylem rays. The vessels are elliptical, thick walled, either in radial multiples or solitary. The fibers are thick walled and lignified. The primary xylem occurs in radial rows around the pith region (Figure 1c).
Figure 1. Microscopy of of T. lampas stems.
A: Transverse section of the stem; B: Transverse section of the stem bark; C: Transverse section of the stem showing secondary xylem. (Pe - Periderm, DR - Dilated ray, Sc - Sclereids, Ph - Phloem, SPh - Secondary phloem, Ve - Vessel, XF - Xylem fibres)
3.3. Powder characteristics
Powder microscopy of the stem of T. lampas showed the presence of lignified long, elongated and also short, wide fibres with centrally consisting of parenchyma cells, polygonal thin walled epidermal cork cells, narrow, cylindrical, vessel elements, cluster and acicular form of calcium oxalate crystals and also presence of oval and rounded starch grains. Chemo-microscopy revealed the presence of starch grains, lignin and calcium oxalate crystals (Figure 2).
Figure 2. Powder Microscopy of of T. lampas stems.
3.4. Physicochemical constants
The ash values, extractive values, moisture content and total crude fibre content of stems were determined. The total ash, acid insoluble, water soluble and sulphated ash values were observed to be (9.03 ± 0.05) % w/w, (1.50 ± 0.01) % w/w, (2.51 ± 0.02) % w/w, (7.50 ± 0.01) % w/w respectively on dry weight basis whereas extractive values of ethanol and water were found to be (0.24 ± 0.02) % w/w and (0.08 ± 0.01) % w/w respectively. The moisture content and total crude fibre content of stem powder were (6.03 ± 0.05) % w/w and (47.36 ± 0.32) % w/w respectively (Table 1).
Table 1. Physicochemical parameters of T. lampas stems.
| Parameters | Mean ± SD (% w/w) |
| Total ash | 9.03 ± 0.05 |
| Acid-insoluble ash | 1.50 ± 0.01 |
| Water-soluble ash | 2.51 ± 0.02 |
| Sulphated ash | 7.50 ± 0.01 |
| Ethanol soluble extractive | 0.24 ± 0.02 |
| Water soluble extractive | 0.08 ± 0.01 |
| Moisture content | 6.03 ± 0.05 |
| Total crude fibre content | 47.36 ± 0.32 |
3.5. Fluorescence characteristic
Fluorescence characteristic of powdered stem of T. lampas were observed for resolution of doubtful specimen. The stem powder of T. lampas was observed in visible light, short and long ultra-violet light (Table 2).
Table 2. Fluorescence analysis of powdered stems of T. lampas.
| Reagents | Color observed in ordinary light | Color observed under ultraviolet light |
|
| Short (254 nm) | Long (365 nm) | ||
| 1 N NaOH in methanol | Brown | Black | Blue |
| 1 N NaOH in water | Brown | Bluish brown | Blue |
| 1 N HCl | Brown | Green | Blue |
| 50% HNO3 | Brown | Black | Brown |
| 50% H2SO4 | Brown | Black | Brown |
3.6. Behaviors of stem powder of T. lampas with different chemical reagent
Behaviors of stem powder of T. lampas with different chemical reagents showed presence of steroids, starch, alkaloid, flavonoids and proteins (Table 3).
Table 3. Behavior analysis of powdered stem of T. lampas.
| Reagents | Colour/ppt | Constituents |
| Powder as such | Brown | - |
| Concentrated H2SO4 | Brownish red | Steroid (+) |
| Aqueous ferric chloride | No black color | Tannins (-) |
| Aqueous iodine solution | Blue color | Starch (+) |
| Aqueous mercuric chloride | Brown | Alkaloid (+) |
| Picric acid | Yellow ppt | Alkaloid (+) |
| Magnesium HCl | Pink | Flavonoids (+) |
| Aqueous silver nitrate solution | No change | Protein (+) |
| Ammonia solution | No change | Anthraquinone glycosides (-) |
| Aqueous KOH | Greenish | Anthraquinone glycosides (-) |
“+”- Present; ““-Absent
3.7. Heavy metal and mineral analysis
ASS analysis of stem powder showed presence of heavy metals namely lead, and minerals such as zinc, copper, irons, nickel and chromium whereas in stem ash showed presence of heavy metals namely lead, aluminum and minerals such as zinc, copper irons, nickel and chromium (Table 4).
Table 4. Heavy metal and mineral content analysis of T. lampas stems.
| Heavy metals and mineral content | Values* in stem ash (ppm) | Values* in stem powder (ppm) |
| Lead | 4.290 | 4.960 |
| Aluminum | 183.000 | N. D. |
| Magnesium | N. D. | N. D. |
| Zinc | 27.000 | 27.300 |
| Copper | 12.600 | 11.600 |
| Iron | 142.000 | 158.000 |
| Nickel | 3.610 | 3.590 |
| Chromium | 0.148 | 0.297 |
N. D. - Not Detected; * - Average of three determinations.
3.8. Extractive values and preliminary phytochemical analysis
The extractive values of petroleum ether, methanolic, ethyl acetate soluble, ethyl acetate insoluble and aqueous extract were found to be 3.75%, 8.0%, 3.8%, 4.2%, 8.44% w/w respectively (Table 5). Preliminary phytochemical analysis mainly revealed the presence of carbohydrates, glycosides, flavonoids, tannins, steroids and proteins (Table 6).
Table 5. Extractive values of T. lampas stem extracts.
| Extract | Yield (% w/w) | Color of extract |
| Petroleum ether | 3.75 | Yellow |
| Methanolic | 8.00 | Reddish brown |
| Ethyl acetate soluble | 3.80 | Reddish brown |
| Ethyl acetate insoluble | 4.20 | Brown |
| Aqueous | 8.44 | Brownish black |
Table 6. Preliminary phytochemical analysis of T. lampas stems extracts.
| Chemical Constituents | Chemical tests | Petroleum ether extract | Methanol extract | Ethyl acetate soluble fraction | Ethyl actate Insoluble fraction | Aqueous extract |
| Alkaloids | Dragendorff's test | _ | _ | _ | _ | _ |
| Mayer's reagent | _ | _ | _ | _ | _ | |
| Carbohydrates | Molisch's test | _ | _ | _ | + | + |
| Barfoed's test | _ | _ | _ | + | + | |
| Glycosides | Borntrager's test | _ | _ | + | + | + |
| Keller-killianin test | _ | + | _ | _ | _ | |
| Saponin glycosides | Foam test | _ | _ | _ | _ | _ |
| Flavonoids | Shinoda test | _ | + | + | + | + |
| Sodium hydroxide test | _ | + | + | + | + | |
| Lead acetate test | _ | + | + | + | + | |
| Tannins | Ferric chloride test | _ | + | + | + | + |
| Phenazone test | _ | + | + | + | + | |
| Steroids | Salkowski test | + | + | _ | _ | _ |
| Libermann-burchard test | + | + | _ | _ | _ | |
| Proteins | Biuret test | _ | _ | _ | _ | + |
+: Present -: Absent.
3.9. GC-MS analysis of saponifiable matter
GC-MS analysis of saponifiable matter of the petroleum ether extract gives the idea about chemical characters of compounds present in the extract. The result showed the presence of fatty acids such as dodecanoic acid (10.31%), tetradecanoic acid (8.85%), n-hexadecanoic acid (28.33%), 9-tetradecenal (7.68%) (Table 7).
Table 7. CG-MS analysis of saponifiable matter of petroleum ether extract of T. lampas stems.
| Compound | Molecular formula | Retention time | Concentration (%) | M+ | Base peak |
| Dodecanoic acid | C12 H24 O2 | 9.134 | 10.31 | 200 | 73 |
| Tetradecanoic acid | C14H28O2 | 11.514 | 8.85 | 228 | 73 |
| n- hexadecanoic acid | CH3(CH2)14COOH | 13.715 | 28.33 | 256 | 73 |
| 9-tetradecenal | C14H26O | 15.524 | 7.68 | 210 | 55 |
3.10. HPTLC fingerprinting of T. lampas stems extracts
In HPTLC fingerprinting, it was observed that one of the constituent of petroleum ether extract appears at Rf 0.35, 0.34, methanolic extract at Rf 0.34, Standard β-sitosterol at Rf 0.34 and ethyl acetate soluble fraction of methanolic extract at Rf 0.35. In another plate, it was also observed that one of the constituent of methanolic extract appears at Rf 0.52, 0.56, ethyl acetate soluble fraction of methanolic extract at Rf 0.52, 0.56 and standard quercetin at Rf 0.55 and aqueous extract at Rf 0.56. Hence from the above observation, β-sitosterol and quercetin might be the constituents of stems of T. lampas (Figure 3).
Figure 3. HPTLC fingerprinting of different extracts of of T. lampas stems.
3A: Chromatogram spraying by 10% H2SO4; 3B: Chromatogram at scanned 254 nm; 3C: Chromatogram at scanned 366 nm; 3D:Chromatogram at daylight; 3E: Chromatogram at scanned 254 nm; 3F: Chromatogram at scanned 366 nm
4. Discussion
Pharmacognostic standardization including physico-chemical evaluation is meant for identification, authentication, detection of adulteration and also compilation of quality control of crude drugs. Since the plant, T. lampas is useful in traditional medicine for the treatment of some ailments, it is important to standardize it for use as a drug. Physico-chemical evaluation used to determine numerical standards reported in this work could be useful for the adulterants resolution of doubtful specimen or improper handling of drugs and compilation of a suitable monograph. The total ash is particularly important in the evaluation of purity of drugs, i.e. the presence or absence of foreign inorganic matter such as metallic salts and/or silica. Alcohol and water-soluble extractive values were determined to find out the amount of water and alcohol soluble components. The moisture content of the drug is not too high, thus it could discourage bacterial, fungi or yeast growth, as the general requirement for moisture content in crude drug is not more than 14% w/w[28]. Fluorescence characteristic is a rapid method for resolution of doubtful specimen. When physical and chemical methods are inadequate, the plant material may be identified from their adulterants on the basis of fluorescence characteristics. Behaviors of the powdered drug with different chemical reagents and preliminary phytochemical analysis are helpful for detection of various phytoconstituents. Heavy metal analysis of powdered drug showed presence very low amount of toxic elements whereas presence of considerable amount of mineral elements of drug may be directly or indirectly helpful in the management of many diseases. All herbals are standardized for active constituents. Extract refers to a concentrated preparation of active constituent of a medicinal herb. The concept of standardized extracts definitely provides a solid platform for scientific validation of herbals. Phytochemical evaluation is one of the tools for the quality assessment of plants, which includes preliminary phytochemical screening, chemo profiling and marker compound analysis using modern analytical techniques. GC-MS detection, has found a variety of analytical uses, which performing quality control analysis in both the pharmaceutical and food product industries[29]. In the last two decades HPTLC method has emerged as an important tool for the qualitative and quantitative phytochemical analysis of herbal drugs and formulations[30]. This analysis is the first step towards understanding the nature of active principles and their detailed phytochemistry.
In conclusion, the present study was undertaken with an aim of pharmacognostic standardistion and preliminary phytochemical evaluation of stems of T. lampas providing useful information, which may help in authenticating the genuine plant along with nature of phytoconstituents present in it. This work could be useful for the adulterants resolution of doubtful specimen and compilation of a suitable monograph for its proper identification.
Acknowledgments
The authors express sincere thanks to Anchrom Laboratory, Mumbai, for providing the best available facility and kind support for this work.
Footnotes
Conflict of interest statement: We declare that we have no conflict of interest.
References
- 1.Nadkarni AK. Indian materia medica. 2nd ed. Mumbai: Bombay Popular Prakashan; 2007. p. 629. [Google Scholar]
- 2.Kirtikar KR, Basu BD. Indian medicinal plants. 2nd ed. Dehradun: International Book Distributors; 2005. pp. 336–337. [Google Scholar]
- 3.CSIR . The wealth of india: a dictionary of indian raw material and industrial products. New Delhi: Council of Scientific and Industrial Research; 2005. p. 218. [Google Scholar]
- 4.Jagtap SD, Deokule SS, Bhosle SV. Some unique ethnomedicinal uses of plants used by the Korku tribe of Amravati district of Maharashtra, India. J Ethnopharmacol. 2006;107:463–469. doi: 10.1016/j.jep.2006.04.002. [DOI] [PubMed] [Google Scholar]
- 5.Tamang G. An ethnobiological study of the tamang people. Our Nature. 2003;1:37–41. [Google Scholar]
- 6.Jayakar B, Sangameswaran B. Anti-diabetic activity of Thespesia lampas Dalz & Gibs on alloxan induced rats. Oriental J Pharma Exp Med. 2008;8(4):349–353. [Google Scholar]
- 7.Sangameswaran B, Balakrishnan BR, Malyadri Y, Kumar M, Balakrishnan N, Jayakar B. Anti-hyperlipidaemic effect of Thespesia lampas Dalz and Gibs on triton induced rats. Res J Pharm Technol. 2008;1(4):533–534. [Google Scholar]
- 8.Sangameswaran B, Chumbhale D, Balakrishnan BR, Jayakar B. Hepatoprotective effects of Thespesia lampas Dalz & Gibs in CCl4 induced liver injury in rats. Dhaka Univ J Pharm Sci. 2008;7(1):11–13. [Google Scholar]
- 9.Kumaraswamy MV, Satish S. Antioxidant and anti-lipoxygenase activity of Thespesia lampas Dalz & Gibs. Advan Biol Res. 2008;2(3–4):56–59. [Google Scholar]
- 10.Sangameswaran B, Balakrishnan BR, Chumbhale D, Jayakar B. In vitro antioxidant activity of roots of Thespesia lampas Dalz and Gibs. Pak J Pharm Sci. 2009;22(4):368–372. [PubMed] [Google Scholar]
- 11.Kosalge SB, Fursule RA. Investigation of in vitro anthelmintic activity of Thespesia lampas (Cav.) Asian J Pharma Clin Res. 2009;2(2):69–71. [Google Scholar]
- 12.Adhikari BS, Babu MM, Saklani PL, Rawat GS. Distribution, use pattern and prospects for conservation of medicinal shrubs in Uttaranchal state. Indian J Mountain Sci. 2007;4:155–180. [Google Scholar]
- 13.Luckefahr MJ, Fryxell PA. Content of gossypol in plants belonging to genera related to cotton. Econ Bot. 1967;21:128. [Google Scholar]
- 14.Valsaraj R, Pushpangadan P, Smitt UW, Adsersen A, Nyman U. Antimicrobial screening of selected medicinal plants from India. J Ethnopharmacol. 1997;58:75–83. doi: 10.1016/s0378-8741(97)00085-8. [DOI] [PubMed] [Google Scholar]
- 15.Kokate CK. Practical pharmacognosy. 4th ed. New Delhi: Vallabh Prakashan; 2010. pp. 17–26. [Google Scholar]
- 16.O'Brien TP, Feder N, Mc Cull ME. Polychromatic staining of plant cell walls by toluidine blue-O. Protoplasma. 1964;59:364–373. [Google Scholar]
- 17.Government of India . The Ayurvedic pharmacopoeia of India. 1st ed. Part I. Vol VI. New Delhi: Ministry of Health and Family Welfare, Department of Indian System of Medicines and Homeopathy; 2009. pp. 242–244. [Google Scholar]
- 18.Khandelwal KR. 19th ed. Pune: Nirali Prakashan; 2008. Practical pharmacognosy – technique and experiments; p. 160. [Google Scholar]
- 19.Pratt RJ, Chase CR. Fluorescence of powdered vegetable drugs with particular reference to development of a system of identification. J Am Pharm Assoc. 1949;38:324–333. doi: 10.1002/jps.3030380612. [DOI] [PubMed] [Google Scholar]
- 20.Kokoski CJ, Kokoski RJ, Slama FJ. Fluorescence of powdered vegetable drugs under with particular reference to devlopment of ultraviolet light radiation. J Am Pharma Assoc. 1958;47:715–717. doi: 10.1002/jps.3030471010. [DOI] [PubMed] [Google Scholar]
- 21.Sing VK, Govil GS. Recent progress in medicinal plants: ethnomedicine and pharmacognosy. Texas: SCI Tech publishing LIC; 2002. p. 325. [Google Scholar]
- 22.Saraswathy A, Devi SN, Ramasamy D. Antioxidant, heavy metals and elemental analysis of Holoptelea integrifolia Planch. Indian J Pharm Sci. 2008;70:683–576. doi: 10.4103/0250-474X.45419. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Sreenivasulu M, Jayaveera KN, Madhusudhana Chetty C, Gnanaprakash K, Adinarayana K. Heavy metal analysis of various parts of Ficus mollis (Vahl) by HPTLC. Int J ChemTech Res. 2010;2:807–812. [Google Scholar]
- 24.Mukherjee PK. Quality control of herbal drugs. 1st ed. New Delhi: Business Horizons Pharmaceutical Publishers; 2008. pp. 379–412. [Google Scholar]
- 25.Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 43th ed. Pune: Nirali Prakashan; 2009. pp. A1–A6. [Google Scholar]
- 26.Government of India . New Delhi: Ministry of Health and Welfare, Controller of Publication; 2007. Indian pharmacopoeia; p. 89. [Google Scholar]
- 27.Sethi PD. Quantitative analysis of pharmaceutical formulations. 1st ed. New Delhi: CBS Publishers and Distributors; 1996. HPTLC; pp. 3–73. [Google Scholar]
- 28.Anonymous . African pharmacopoeia. 1st ed. Lago: Organisation of African Unity/The Scientific, Technical and Research Commission (OAU/STRC); 1986. p. p.123. [Google Scholar]
- 29.Smith DC, Forland S, Bachanos E, Matejka M, Barrett V. Qualitative analysis of citrus fruit extracts by GC/MS. Chem Educator. 2001;6:28–31. [Google Scholar]
- 30.Sajeeth CI, Mann PK, Manavalan R, Jolly CI. Quantitative estimation of gallic acid, rutin and quercetin in certain herbal plants by HPTLC method. Der Chimica Sinica. 2010;1:80–85. [Google Scholar]