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During the present study HPTLC analysis of Solanum nigrum, a highly medicinal plant
has been carried out. Simultaneous determination of lupeol and β-sitosterol was
carried out on different accessions of the plant. The leaves of the plant among
all the four accessions have been found to be rich in lupeol while the stem
samples were found to be rich in β-sitosterol.
Keywords: Solanum nigrum, Lupeol, β-sitosterol,
HPTLC
INTRODUCTION
S. nigrum is a widely used plant in oriental medicine.
It is used in hepatitis, fever, dysentery and bowel disorders. The juice of the
plant is used on ulcers and other skin diseases. The fruits are used as a
tonic, laxative, appetite stimulant and are also used for treating asthma and
diabetes. The juice from its roots is used against asthma and whooping cough.
Apart, the plant is considered to be anti-tumorigenic, anti-oxidant,
anti-inflammatory, hepatoprotective, diuretic and antipyretic activity.
Berries are used in fevers, diarrhoea, eye diseases and hydrophobia.
Juice of the plant is hydragogue cathartic, diuretic, in blood spitting, piles,
in enlargement of liver, dysentery, etc. Young shoots are used in treating skin
diseases and psoriasis [1]. The plant is a traditional remedy for hepatitis,
fever, ulcer and various immunological applications in cancer and others. The
plant is beneficial in preventing hepatotoxicity and cytotoxicity thus
improving functions of liver and kidney. It also finds use in analgesic,
anti-inflammatory, antimicrobial, anti-diabetic, immunostimulant, central
nervous system and brain functioning. It can really contribute to medical and
pharmaceutical practices [2].
S. nigrum is reported to have many
glycoalkaloids like solanine, salsodin, solamajine, solamargine, chaconine,
saponins, etc. Out of which solanine, salsodin and chaconine are more
commercially exploited. It has been reported that the plant leaves contain the
highest concentration of gentisic acid, luteolin, apigenin, kaempferol and
m-coumaric acid [3]. The plant has also been reported to contain
(+)-pinoresinol, (+)-syringaresinol, (+)-medioresinol, scopoletin,
tetracosanoic acid and beta-sitosterol [4]. Therefore, four accessions of S. nigrum L. (S1, S2,
S3 and S4) were selected for estimation of lupeol and
β-sitosterol.
MATERIALS AND
METHODS
In the present study, different parts of the collected plants were
subjected to phytochemical screening of pharmacologically important compounds.
The plants were air dried, powered in grinder and were stored at room
temperature. The powdered plant parts of each sample were dissolved in 20 ml of
aqueous methanol for overnight. The extract was concentrated and dried using
rotary evaporator under reduced pressure. 2 mg of each dried extract was again
dissolved in 2 ml of methanol to obtain 1 mg/1 ml concentration and stored at
4°C till further analysis.
HPTLC
instrumentation and conditions
Concentration range from 200-10,000 mg of standard solutions were
spotted on silica gel 60 F254 HPTLC plate (Merck, India) using CAMAG Linomat Ⅴ
automatic spotter (Dosage speed: 150 nL/s, Syringe size: 100 µL, Band length:
6.0 mm). Plates were developed in a twin-through chamber (20 × 10 cm) to a
distance up to 8 cm. The data regarding the bioactive markers used, composition
of the solvent system, derivatizing reagent used and wavelength of the entire
marker compounds. After development, the plates were first air dried and then
oven dried at 105°C for 3-4 min. Further, the
plates were derivatized with p-anisaldehyde
sulphuric
RESULTS AND
DISCUSSION
In the present study, the leaf regions of the
plant among all the four accessions (S1, S2, S3
and S4) have been found to be rich in lupeol while the reverse trend
has been observed in case of stem regions, i.e., the stem region has been found
to be rich in β-sitosterol. Otherwise the maximum amount of lupeol has been
observed to be present in leaf sample of S1 while that of
β-sitosterol was observed in root sample of S3. Among the young and
mature fruit samples of S2, S3 and S4, the
amount of β-sitosterol has been observed to be more in young fruits than the
mature ones in both S2 and S3 but the opposite of this
has been observed in case of S4.
A mixture of Toluene:Ethyl acetate:Glacial
Acetic Acid (14.5:4.5:1.0 v/v/v) was used as mobile phase for the separation of
lupeol. The compound is detected at 525 nm (Figure
2) with Rf value 0.61 (Figure
3 and Table 1). HPTLC densitometric chromatogram of standard tracks and
sample tracks of four accessions of S.
nigrum L. has been given in Figures
1 and 2.
Lupeol was not detected in mature fruit
samples of S2 and in root samples of both S3 and S4.
Among the leaf samples of all the four accessions of S. nigrum L. maximum concentration of Lupeol was detected in S1
leaf sample, followed by S2 leaf sample, S3 leaf sample,
and S4 leaf sample. Similarly, among the stem samples of the studied
accessions, maximum amount has been detected in S1 stem sample,
followed by S2 stem sample, S4 stem samples and S3
stem samples. Since Lupeol was not detected in the mature fruit sample of S2
therefore, the maximum concentration of Lupeol among rest of the mature fruit
samples was found to be maximum in S4 mature fruit sample, followed
by S3 mature fruit sample and S1 mature fruit sample
while among the raw fruit samples of S2, S3 and S4,
maximum concentration of Lupeol was detected in S3, followed by S4
and S2 (Table 2).
Separation of β-sitosterol has been done
using Toluene: Ethyl acetate: Glacial Acetic Acid (14.5: 4.5: 1.0 v/v/v) as
mobile phase. The bands were visualised at 525 nm (Figures 1 and 2). The Rf value of β-sitosterol is observed to be
0.54 (Table 1). HPTLC densitometric
chromatogram of standard tracks and sample tracks of four accessions of S. nigrum L. has been given in Figure 2. In the present studies,
β-sitosterol was detected in all plant parts of presently studied four
accessions. Maximum amount of β-sitosterol was detected in S3 root
sample (60.38 ± 0.51 mg/g of DW), followed by S1 stem sample, S3
young fruit sample, S4 root sample, S2 stem sample, S4
stem sample, S4 mature fruit sample, S2 root sample, S4
young fruit sample, S1 leaf sample, S2 young fruit
sample, S3 mature fruit sample, S1 mature fruit sample, S2
mature fruit sample, S2 leaf sample, S3 stem sample, S4
leaf sample and the least was detected in S3 leaf sample (0.36 ±
0.21 mg/g of DW).
Among the leaf samples, maximum amount of
β-sitosterol has been found to be present in S1, followed by S2,
S4 and S3 while among the stem samples the highest
concentration of β-sitosterol in the present study has been recorded in S1,
followed by S2, S4 and S3. The concentration
of β-sitosterol in the root samples of S2, S3 and S4
has been observed to be present in enormous amount with maximum in S3
root sample, followed by S4 root sample and S2 root
sample. Similarly, in young fruit samples, the highest concentration of
β-sitosterol has been recorded in S3, intermediate in S4
and the lowest being detected in S2 while among the mature fruit
samples, maximum concentration of β-sitosterol has been found to be present in
S4, followed by S3, S1 and S2 (Table 2). Overall, lowest amount of
β-sitosterol has been observed to be present in the leaf sample of the plant
among all the four accessions. The maximum amount of lupeol has been observed
to be present in leaf sample of S1, while that of β-sitosterol was
observed in root sample of S3. Among the young and mature fruit
samples of S2, S3 and S4, the amount of
β-sitosterol has been observed to be more in young fruits than the mature
fruits in both S2 and S3 but the opposite of this has
been observed in case of S4 where mature fruit sample contained more
amount of β-sitosterol than the young fruit sample (Figure 3).
Earlier, Jagtap et al. [5] carried out
pharmacognostic and phytochemical investigation of root of S. nigrum Linn. The preliminary phytochemical screening of three
extracts of the root powder revealed the presence of alkaloids, tannins and
saponins along with other phytoconstituents and HPTLC studies, the alcoholic
extract showed presence of ten and seven phytoconstituents at 254 nm and 366
nm.
ACKNOWLEDGMENT
This study was funded by Department of Biotechnology (DBT), New Delhi, DBT-IPLS Project with reference number BT/PR 4548/NF/22/146/2012 to Dr. Ramanpreet. The authors are also thankful to Head, Department of Botany, Punjabi University, Patiala, for all the necessary laboratory facilities.
1. Chopra
RN, Nayar SL, Chopra IC, Asolkar LV, Kakkar KK, Chakre OJ, et al. (1956)
Glossary of Indian medicinal plants. Council of Scientific and Industrial
Research, New Delhi.
2. Nyeem
MAB, Rashid AMU, Nowrose M, Hossain MA (2017) Solanum nigrum (Maku): A review of pharmacological activities and
clinical effects. IJAR 3: 12-17.
3. Huang
HC, Syu KY, Lin JK (2010) Chemical composition of Solanum nigrum Linn extract and induction of autophagy by leaf
water extract and its major flavonoids in AU565 breast cancer cells. J Agric
Food Chem 58: 8699-8708.
4. Zhao
Y, Liu F, Lou HX (2010) Studies on the chemical constituents of Solanum nigrum. Zhong yao cai 33:
555-556.
5. Jagtap
CY, Prajapati PK, Rudrappa HC, Shukla VJ (2013) Pharmacognostic and
phytochemical investigation of root of Solanum
nigrum Linn.: An ethnomedicinally important herb. Int J Green Pharm 7:
46-49.
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