Pharmacognostic Evaluation and HPTLC Finger Printing of Rhizome of Chlorophytum borivilianum Sant. and F. from Nepal

Copyright © 2018 Phcog.Net. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. ABSTRACT Introduction: Chlorophytum borivilianum Sant. and F.; commonly known as Shveta Musali from the family Liliaceae is a perennial herb. It is used in Ayurveda, Traditional Chinese Medicine, Unani and in folklore practice as an aphrodisiac herb. Present study depicts pharmacognostic features of Chlorophytum borivilianum Sant. and F. collected from Nepal. Methods: Macromicroscopic analyses, physico-chemical studies and HPTLC finger printing of rhizomes of Chlorophytum borivilianum Sant. and F. were carried out according to pharmacopoeial procedures. Results: Microscopic analysis has shown presence of epidermis, cork, cortex, collenchymatous cells, starch grains, cluster crystals of calcium oxalate, idioblast, phloem, vascular bundles, pitted xylem parenchyma, sclereids, stone cells, fragment of epiblema, and acicular needles. Preliminary phytochemical analysis revealed presence of alkaloid, carbohydrate, carboxylic acid, resins and saponins. TLC photo-documentation revealed presence of many phyto-constituents with different Rf values and HPTLC densitometric scan of the plates showed numerous bands under short UV, long UV and 620 nm (after derivatisation). Conclusion: Chlorophytum borivilianum Sant. and F. was evaluated for its pharmacognostic features and HPTLC. These specific identities will be useful in identification and authentication of the raw drug.


INTRODUCTION
Chlorophytum includes nearly 300 species which are distributed throughout tropical and subtropical parts of the world.Seventeen species of Chlorophytum is recorded in India. 1 Among them Chlorophytum borivilianum Sant.and F. is having highest saponin content which is responsible for its therapeutic utilities. 2It was first reported in India in 1954 and reached rare status in nature due to over exploitation. 3C. arundinaceum Baker, C. laxum R.Br., C. tuberosum Baker, C. orchidastrum Hook.f., p.p. non Lindl.are used as adulterant and substitutes. 4C. borivilianum is commercially cultivated and commonly used by pharmaceuticals. 5hlorophytum borivilianum Sant.and F., also known as Shveta Musali, from the family Liliaceae, is a perennial herb, 10-35 cm in height; rhizome elongated, cylindrical, fleshy.Leaves are basal, linear-lanceolate and membranous with short petioles.7][8] It is used in Ayurveda, Traditional Chinese Medicine, Unani and in folklore practice as an aphrodisiac herb.It's rhizome is Shukrala, Rasayana, Vrisya, Balya, Brimhana, Madhura, Tikta, Snigdha, Sheeta, and Laghu. 4,9,106]

Plant material
Fresh rhizomes were collected from Chitwan District, Nepal in the month of November -December.The plant material was authenticated at Department of Dravyaguna, SDM College of Ayurveda Hassan, Karnataka and the voucher specimen was deposited in the respective herbarium for future reference (SDMCAH-DG/2017/14).The rhizomes were cleaned and shade dried.The dried rhizomes were coarsely powdered and used for macroscopic, microscopic characterization, phyto-chemical analysis and HPTLC.

Organoleptic and Macroscopic Evaluation
Fresh and dried rhizomes along with the powder were evaluated for their organoleptic and macroscopic features i.e. size, shape, color, odor, taste, texture and specific botanical characters were evaluated as per the standard procedure. 17The external features of the test samples were documented using Canon IXUS digital camera.

Microscopic evaluation
Transverse section of rhizome: Sample was preserved in fixative solution.The fixative used was FAA (Formalin-5ml + Acetic acid-5ml + 70% Ethyl alcohol-90ml).9] The preserved specimens were cut into thin transverse section using a sharp blade and the sections were stained with safranin.The slides were also stained with iodine in potassium iodide for detection of starch.Transverse sections were photographed using Zeiss AXIO trinocular microscope attached with Zeiss Axio Cam camera under bright field light.Magnifications of the figures are indicated by the scale-bars.

Powder microscopy
Pinch of powder of rhizome previously sieved was put on the slide and mounted in glycerin.Powder characters are observed under the Zeiss AXIO trinocular microscope attached with Zeiss Axio Cam camera under bright field light.

Physico-chemical analysis
Physico-chemical parameters viz.loss on drying at 105ºC, total ash, acid insoluble ash, water soluble ash, ethanol and water soluble extractive values were evaluated using standard methods. 20eliminary phyto-chemical screening Ethanolic extract of C. borivilianum Sant.and F. was subjected to qualitative evaluation for the presence or absence different groups of phytoconstituents such as alkaloids, flavonoids, saponins, carbohydrates, carboxylic acid, coumarins, phenol, quinine, resins, steroid, tannin, terpenoid, and amino acids. 20Detail of phyto-chemical evaluation is illustrated in Table 3.

HPTLC finger printing
One gm of powdered sample of Chlorophytum borivilianum Sant.and F. was suspended in 10.0ml of alcohol (99.9%) with intermittent shaking for the first few hours and kept for 24 h at room temperature followed by filtration, made up to 10.0ml with ethanol; 8µl of the above extract was applied on a pre-coated silica gel F254 on aluminum plates to a band width of 7 mm using Linomat 5 TLC applicator.The plate was developed in Toluene: Ethyl acetate (7.0: 3.0).The developed plates were visualized in short UV, long UV and then derivatised with vanillin sulphuric acid and scanned under UV 254nm, 366nm and 620nm.Retention factor (Rf), color of the spots and densitometric scan were recorded using CAMAG Scanner 4. [21][22]

Organoleptic and macroscopic observations
Results obtained from organoleptic and macroscopic observations of fresh rhizome and powder are illustrated in the Table 1.

Microscopic evaluation
Microscopic evaluation of transverse section of rhizome of Chlorophytum borivilianum Sant.and F. revealed the presence of epidermis, cork, starch grains, idioblast cells, outer cortex, xylem, phloem, radial vascular bundle, collenchyma cells, single layered endodermis, stellar region, pith, and numerous cluster crystals.The details of the microscopic evaluation of transverse section of rhizome are presented in Figure 1.Microscopic  evaluation of powder of rhizome of Chlorophytum borivilianum Sant.and F. revealed the presence of starch grains, pitted xylem parenchyma, vessels, cork, sclereids, stone cells, fragment of epiblema, acicular needles.The details of the powder microscopy of rhizome are presented in Figure 2.

Physico-chemical analysis
Physico-chemical characters were evaluated and the results obtained are illustrated in Table 2.

Phytochemical Evaluation
Phytochemical evaluation of Chlorophytum borivilianum Sant.and F. revealed the presence of alkaloid, carbohydrate, carboxylic acid, resins and saponins in the rhizome.Detail of phyto-chemical evaluation is illustrated in Table 3.

HPTLC Finger printing
R f values and color of the spots in chromatogram developed in toluene: ethyl acetate (7.0:3.0) for ethanolic extract of rhizomes of Chlorophytum borivilianum Sant.and F. were recorded.The details of Rf value is given in Table 4. TLC photo-documentation revealed presence of many phytoconstituents with different Rf values and HPTLC densitometric scan of the plates showed numerous bands under short UV, long UV and 620 nm (after derivatisation).

Macroscopic characters
Rhizome of C. borivilianum was elongated, with whitish external surface.It was smooth and having horizontal wrinkles on drying.It was not having any characteristics odor.It was having Madhura (Sweet), Tikta (Bitter) and mucilaginous taste.Powder of C. borivilianum was brownish in color.

Microscopic characters
Transverse section of rhizome of C. borivilianum was having epidermis, cork, starch grains, idioblast cells, outer cortex, xylem, phloem, radial vascular bundle, collenchyma cells, single layered endodermis, stellar region, pith, and numerous cluster crystals.Powder microscopy of the given sample had pitted xylem parenchyma, starch grains, cork cells, sclereids, bundle of fibers, vessels, acicular needles, fragments of epiblema and stone cells.
Relevance of finding from current study related to microscopic evaluation can be also substantiated from the earlier works. 23

Phytochemical Analysis
Phytochemical analysis of Chlorophytum borivilianum Sant.and F. had shown presence of alkaloid, carbohydrate, carboxylic acid, resins and saponins.Previous studies had shown presence of steroid, glycosides, saponins, and triterpenoid starch in Chlorophytum borivilianum Sant.and F. [23][24] TLC AND HPTLC R f values and color of the spots in chromatogram developed in toluene: ethyl acetate (7.0:3.0) for ethanolic extract of rhizomes of Chlorophytum borivilianum Sant.and F. were recorded.TLC photo-documentation revealed presence of many phytoconstituents with different Rf values and HPTLC densitometric scan of the plates showed numerous bands under short UV, long UV and 620 nm (after derivatisation).Photo documentation (solvent system pet ether: ether acetate)

Table 2 : Physico-chemical Evaluation of Rhizome of Chlorophytum boriv- ilianum Sant. and F.
Total eight spots detected in Chlorophytum borivilianum Sant.and F in different Rf value.Number of spots indicates the total number of active chemical components present in the given sample.Chlorophytum borivilianum Sant.and F. has been carried out as per pharmacopoeial methodology.The detail presented in the study shows the qualitative presence of various secondary metabolites and in the rhizome of Chlorophytum borivilianum Sant.and F. Thus, the study was helpful in the qualitative analysis of genuinity of the given drug.