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

Adhikari, Anuradha, and Suchitra: Pharmacognostic Evaluation and HPTLC Finger Printing of Rhizome of Chlorophytum borivilianum Sant. and F. from Nepal

Authors

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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.2 It was first reported in India in 1954 and reached rare status in nature due to over exploitation.3 C. arundinaceum Baker, C. laxum R.Br., C. tuberosum Baker, C. orchidastrum Hook.f., p.p. non Lindl. are used as adulterant and substitutes.4 C. borivilianum is commercially cultivated and commonly used by pharmaceuticals.5 Chlorophytum 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. White flowers with six petals, small, black seeds enclosed in flowering boles.68 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.4,9,10 It has shown spermatogenic, aphrodisiac, immune-modulatory, anti-diabetic, anti-oxidant, anti-stress, anti-microbial, anti-aging, anti-tumor and anti-inflammatory activities.1115 Saponins (borivilianosides E-H), flavonoids, proteins, carbohydrate, phenolics, triterpenoids, tannis, sucrose, glucose, fructose, galactose, mannose and xylose have been reported from C. borivilianum.2-3,1516

MATERIALS AND METHODS

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.17 The 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). The materials were left in FAA for more than 48 h.1819 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.20

Preliminary phyto-chemical screening

Ethanolic extract of C. borivilianum Sant. and F. was subjected to qualitative evaluation for the presence or absence different groups of phyto-constituents such as alkaloids, flavonoids, saponins, carbohydrates, carboxylic acid, coumarins, phenol, quinine, resins, steroid, tannin, terpenoid, and amino acids.20 Detail 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 4.2122

RESULTS

Organoleptic and macroscopic observations

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

Table 1

Organoleptic and Macroscopic Evaluation of Chlorophytum borivilianum Sant. and F.

ObservationsRhizomePowder
Size5-12cm long, 1.5-2cm diameterNA
ShapeElongatedNA
ColorWhitishBrownish
OdorNot characteristicsNot characteristics
TasteMadhura, Tikta, MucilagenousMadhura, Tikta, Mucilagenous
TextureSmooth and having horizontal wrinkles on dryingSmooth

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.

Figure 1

Macro-microscopic features of Rhizome of Chlorophytum borivilianum Sant. and F.

Fig. la - Rhi/ome; Fig. lb - Powder; Fig. Ic - TS through outer region; Fig. Id - Enlarged TS through outer region; Fig. le - TS through inner region; Fig. If - Enlarged xylem and phloem; E - Epidermis; Ck - Cork; Id - Idioblast; OC - Outer cortex; ACr - Acicular crystal; SG - Starch grain: Col - Collenchyma: End - Endodermis; IC - lnner cortex; Pi - Pith; Xy - Xylem; Ph - Phloem.

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Figure 2

Powder Microscopy of Rhizome of Chlorophytum borivilianum Sant. and F.

Fig: 2a - Parenchyma with starch grains; Fig: 2b - Parenchyma with bunch of starch grains; Fig: 2c - Isolated starch: Fig: 2d - Fragments of Epiblema: Fig: 2e - Vessels; Fig: 2f - Bundle of fibres; Fig: 2g - Cork cells; Fig: 2h - Group of Stone cells: Fig: 2i - Pitted xylem parenchyma; Fig: 2j - Group of Sclereids; Fig: 2k - Isolated sclereids; Fig: 21 - Aciculur needles,

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Physico-chemical analysis

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

Table 2

Physico-chemical Evaluation of Rhizome of Chlorophytum borivilianum Sant. and F.

ObservationsObserved values
Foreign matter7.93±0.06%
Loss on drying8.70±0.02%
Total ash8.91±0.05%
Acid insoluble ash4.51±0.01%
Water soluble ash value06.45±0.02%
Alcohol soluble extractive3.97±0.04%
Water soluble extractive24.78±0.11%

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.

Table 3

Phytochemical evaluation of Chlorophytum borivilianum Sant. and F.

 TestColor if positiveResultRemarks
1.Alkaloids   
 Dragendrof‘s testOrange precipitateOrange precipitatePresent
 Wagners testRed precipitateRed precipitatePresent
 Mayers testDull white precipitateDull white precipitatePresent
2.Steroids
 Liebermann- buchard testBluish greenNo Bluish greenAbsent
 Salkowski testBluish red to cherry redRed color in the chloroformAbsent
3.Carbohydrate
 Molish testViolet ringViolet ringPresent
 Fehlings testBrick red precipitateBluish colorAbsent
 Benedicts testRed precipitateBluish colorAbsent
4.Tannin
 With FeCl3Dark blue or green or brownGolden Yellow ColorAbsent
5.Flavanoids
 Shinoda‘s testRed to pinkGolden Yellow ColorAbsent
6.Saponins
 With NaHCO3Stable frothStable frothPresent
7.Triterpenoids
 Tin and thionyl chloride testRedBlack colorAbsent
8.Coumarins
 With 2 N NaOHYellowColorless solutionAbsent
9.Phenols
 With alcoholic ferric chlorideBlue to blue black, brownGolden Color solutionAbsent
10.Carboxylic acid
 With water and NaHCO3Brisk effervescenceBrisk EffervescencePresent
11.Resin
 With aqueous acetoneTurbidityTurbidityPresent
12.Quinone
 5% NaOHPink/purple/redColor less solutionAbsent
13.Amino acids
 Ninhydrine reagentPurple colorColor less solutionAbsent

HPTLC Finger printing

Rf 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).

Table 4

Rf values of Ethanolic Extract of Rhizome of Chlorophytum borivilianum Sant. and F.

Short UVLong UVAfter derivatisation
-0.04 (FL. blue)-
--0.24 (L. purple)
---
---
-0.38 (FL. blue)-
---
---
--0.59 (L. purple)
---
0.70 (L. green)--
-0.72 (FD. blue)-
0.77 (L. green)-0.77 (L. purple)
-0.84 (FD. blue)-
D-Dark; L-Light; F- Fluorescent

PHOTO DOCUMENTATION (SOLVENT SYSTEM PET ETHER: ETHER ACETATE)

Two spots were detected under short UV - 254 nm in Chlorophytum borivilianum Sant. and F. (Rf 0.70, 0.77). All of them were having light green color. Four spots were detected under long UV - 366 nm (Rf 0.04, 0.38 – fluorescent light blue color, Rf 0.72, 0.84 – fluorescent dark blue color). Three spots were detected after derivatisation - 620 nm (Rf 0.24, 0.54, 0.77 – light purple color). Detail of HPTLC photo documentation are illustrated in Figure 3.

Figure 3

HPTLC Photo Documentation of Rhizome of Chlorophytum borivilianum Sant. and F.

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HPTLC DENSITOMETRIC SCAN

Total 17 numbers of active components were detected in Chlorophytum borivilianum Sant. and F. having Rf value (0.01, 0.04, 0.25, 0.27, 0.33, 0.39, 0.60, 0.79, 0.85, 0.05, 0.42, 0.79, 0.03, 0.21, 0.28, 0.56, 0.68, 0.86). The detail of HPTLC photo documentation are illustrated in Figure 4. Nine peaks were detected under short UV - 254 nm; among them maximum percentage of area were occupied by Rf 0.01 (35.26%), 0.04 (12.94%), 0.33 (11.11%). Four peaks were detected under long UV - 366nm; among them maximum percentage of area were occupied by Rf 0.01 (67.65%), 0.05 (21.20%), 0.79 (7.54%). Six peaks were detected after derivatization - 620nm; among them maximum percentage of area were occupied by Rf 0.03 (65.06%), 0.68 (13.38%), 0.86 (9.51%).

Figure 4

Densitometric scan of Rhizome of Chlorophytum borivilianum Sant. and F.

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DISCUSSION

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.2324

TLC AND HPTLC

Rf 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)

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.

HPTLC Densitometric scan

Total 17 numbers of active components were detected in Chlorophytum borivilianum Sant. and F. having Rf value (0.01, 0.04, 0.25, 0.27, 0.33, 0.39, 0.60, 0.79, 0.85, 0.05, 0.42, 0.79, 0.03, 0.21, 0.28, 0.56, 0.68, 0.86) among them maximum percentage of area were occupied by Rf 0.01 (35.26% - 254nm); 0.01 (67.65% - 366nm), 0.03 (65.06% - 620nm). Relevance of finding from current study related to HPTLC fingerprinting can be also substantiated from the earlier works.23

CONCLUSION

Macro-microscopic, physico-chemical, preliminary phytochemical and HPTLC finger printing of 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.

ACKNOWLEDGEMENT

Authors are highly grateful to Department of Dravyaguna, SDM College of Ayurveda, Hassan; SDM Center for Research in Ayurveda and Allied science, Udupi for providing the lab facilities.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ABBREVIATIONS

TLC

Thin layer chromatography

HPTLC

High Performance Thin layer chromatography

Rf

Retention factor

UV

Ultra violet

nm

Nano meter

ml

Mili-liter

µl

Micro-liter

viz

Namely.

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PICTORIAL ABSTRACT

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