J Plant Biotechnol 2022; 49(3): 230-239
Published online September 30, 2022
https://doi.org/10.5010/JPB.2022.49.3.230
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: preethahemanth@yahoo.com
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Holostemma annulare (Family Asclepiadaceae) is an invaluable vulnerable medicinal plant; the root tubers are used in Ayurveda medicine and by folk healers to treat various ailments. In this study, Schenk and Hildebrandt medium fortified with the cytokinins 6-benzyl adenine, kinetin, and auxins, including indole 3-butyric acid, indole 3-acetic acid, α-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid, were checked for their efficiency on root tuber induction from different explants. Adventitious root tubers were more successfully induced from in vitro leaf segments and shoots when cultured in Schenk and Hildebrandt medium supplemented with 0.5 mg/l of α-naphthaleneacetic acid. In addition, preliminary phytochemical analysis of in vitro root tubers and identification of different secondary metabolites were conducted. Thin layer chromatography and high performance thin layer chromatography analysis of the crude methanolic extracts of the in vitro root tuber identified the presence of lupeol, a bioactive triterpene. Adventitious root tuber induction offers a novel method for the in vitro production of bioactive metabolites that can be scaled up by bioreactors, thus ensuring the conservation and sustainable utilization of H. annulare. The study warrants further scale-up production and pharmacological investigation that can be extended for pharmaceutical needs.
Keywords Adventitious in vitro root tubers, Holostemma annulare, high performance thin layer chromatography (HPTLC), lupeol, Schenk and Hildebrandt (SH) medium
The tuberous roots of
Explants like nodes and shoot tips collected from disease-free plants of
The
Adventitious roots induced were subcultured to liquid SH medium containing the growth regulator that evoked better induction of
For detecting the presence of bioactive metabolites in the
Chromatographic separation of bioactive component
About 2.25 g
HPTLC analysis of methanolic extracts of root samples (used for TLC separation) were carried out in HPTLC system (CAMAG, Switzerland), CAMAG twin-trough plate development chamber, using silica gel plates (60F254 Manufacturer E. MERCK KGaA). Sample application was carried out on CAMAG Linomat 5 instrument (CAMAG TLC Scanner 3, CAMAG Reprostar 3 photodocument system and WinCATS Software 4.03). The submitted samples are applied to a Silica gel 60 F254 TLC plate (E. Merck, Germany) (10 cm × 10 cm) using Linomat V sample applicator as 8 mm wide bands along with the standard lupeol (1 mg lupeol dissolved in 1 ml chloroform). It is then developed up to 80 mm in a twin trough glass chamber using the Mobile Phase: 16:4, hexane : ethyl acetate. The plate is then derivatised using anisaldehyde sulphuric acid reagent, heated at 110°C for 10 min. The plate is then scanned densitometrically at 580 nm using TLC Scanner 3 equipped with WinCats software. The derivatised plate is then photo documented using CAMAG reprostar 3.
Each
When the effect of different medium formulations such as MS, ½ MS and SH media were tested for shoot culture initiation in
Table 1 . Effect of plant growth regulators in Schenk and Hildebrandt medium on shoot culture initiation in
BA | Kinetin | NAA | Number of shoots | Length of shoots (cm) | Number of nodes per shoots | |||
---|---|---|---|---|---|---|---|---|
Node | Shoot tip | Node | Shoot tip | Node | Shoot tip | |||
0.25 | - | - | 1.88 ± 0.01b | 1.83 ± 0.44b | 11.91 ± 0.01b | 10.21 ± 0.21b | 5.51 ± 0.31b | 5.83 ± 0.10b |
0.5 | - | - | 1.95 ± 0.12a | 1.92 ± 0.21a | 10.82 ± 0.02c | 10.01 ± 0.43b | 6.31 ± 0.31a | 6.16 ± 0.05a |
1.0 | - | - | 1.45 ± 0.37d | 1.56 ± 0.42e | 7.38 ± 0.01f | 8.21 ± 0.31d | 5.91 ± 0.43b | 5.71 ± 0.03b |
2.0 | - | - | 1.34 ± 0.36e | 1.36 ± 0.16f | 6.21 ± 0.01g | 6.41 ± 0.36f | 5.12 ± 0.22b | 5.13 ± 0.11b |
0.25 | 0.5 | - | 1.67 ± 0.43c | 1.66 ± 0.21d | 10.52 ± 0.02c | 9.22 ± 0.41c | 3.92 ± 0.32e | 2.63 ± 0.22e |
0.5 | 0.5 | - | 1.24 ± 0.32f | 1.77 ± 0.43c | 8.23 ± 0.03e | 8.03 ± 0.26d | 3.22 ± 0.23e | 3.34 ± 0.04d |
1.0 | 0.5 | - | 1.13 ± 0.04g | 1.15 ± 0.09g | 6.24 ± 0.04g | 6.14 ± 0.13f | 3.22 ± 0.22e | 3.23 ± 0.13d |
2.0 | 0.5 | - | 1.04 ± 0.08h | 1.05 ± 0.05h | 4.03 ± 0.02h | 3.92 ± 0.02g | 2.82 ± 0.41f | 2.74 ± 0.33e |
0.25 | - | 0.1 | 1.97 ± 0.21a | 1.95 ± 0.03a | 13.72 ± 0.02a | 12.83 ± 0.13a | 6.52 ± 0.28a | 6.43 ± 0.42a |
0.5 | - | 0.5 | 1.85 ± 0.42b | 1.73 ± 0.07c | 9.52 ± 0.02d | 9.33 ± 0.23c | 4.34 ± 0.34d | 4.23 ± 0.17c |
1.0 | - | 1.0 | 1.64 ± 0.11c | 1.54 ± 0.20e | 8.43 ± 0.03e | 8.23 ± 0.25d | 4.12 ± 0.13d | 4.33 ± 0.21c |
2.0 | - | 1.5 | 1.22 ± 0.32f | 1.34 ± 0.72f | 7.93 ± 0.03f | 7.53 ± 0.28e | 3.22 ± 0.15e | 3.32 ± 0.32d |
BA - 6-benzyl adenine; NAA - α-naphthaleneacetic acid
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05).
The initiated shoots subcultured to SH medium supplemented with 2.0 mg/l BA in combination with 0.5 mg/l Kinetin and 1.0 mg/l IBA evoked maximum 7.16 ± 0.15 shoots with a mean length of 6.53 ± 0.03 cm and 4.53 ± 0.05 nodes per shoot (Fig. 1d) (Table 2). Likewise, Gayathri et al. (2009) reported multiple shoot induction in
Table 2 . Effect of plant growth regulators in Schenk and Hildebrandt medium on shoot multiplication in
BA | Kinetin | IBA | NAA | Number of shoots | Length of shoots (cm) | Number of nodes per shoots |
---|---|---|---|---|---|---|
1.0 | 0.5 | 2.13 ± 0.15f | 2.21 ± 0.31e | 2.06 ± 0.05c | ||
2.0 | 0.5 | - | - | 2.23 ± 0.25f | 2.11 ± 0.20e | 1.62 ± 0.41d |
2.0 | 1.0 | - | - | 3.14 ± 0.10e | 2.52 ± 0.24e | 2.23 ± 0.10c |
1.0 | - | 0.5 | 4.23 ± 0.20d | 3.21 ± 0.01d | 2.63 ± 0.25c | |
2.0 | - | 0.5 | - | 4.06 ± 0.20d | 2.42 ± 0.02e | 3.13 ± 0.11b |
2.0 | - | 1.0 | - | 3.36 ± 0.35e | 2.82 ± 0.02e | 3.43 ± 0.20b |
1.0 | - | - | 0.5 | 4.26 ± 0.20d | 1.23 ± 0.03f | 2.41 ± 0.36c |
2.0 | - | - | 0.5 | 3.33 ± 0.28e | 1.81 ± 0.10f | 2.44 ± 0.05c |
2.0 | - | - | 1.0 | 3.1 ± 0.100e | 2.07 ± 0.06e | 2.32 ± 0.10c |
1.0 | 0.5 | 0.5 | - | 4.06 ± 0.05d | 5.23 ± 0.03b | 3.43 ± 0.32b |
2.0 | 0.5 | 0.5 | - | 6.13 ± 0.15b | 6.33 ± 0.25a | 4.46 ± 0.28a |
2.0 | 0.5 | 1.0 | - | 7.16 ± 0.15a | 6.53 ± 0.03a | 4.53 ± 0.05a |
1.0 | 0.5 | - | 0.5 | 4.06 ± 0.05d | 4.23 ± 0.15c | 3.12 ± 0.10b |
2.0 | 0.5 | - | 1.0 | 5.16 ± 0.15c | 5.33 ± 0.13b | 3.16 ± 0.20b |
2.0 | 1.0 | - | 1.0 | 3.13 ± 0.12e | 4.19 ± 0.06c | 2.33 ± 0.30c |
BA - 6-benzyl adenine; IBA - indole 3-butyric acid; NAA - α-naphthaleneacetic acid
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05).
Better root tuber induction devoid of callusing was noticed in SH medium containing 0.5 mg/l NAA (Fig. 1e-g) that produced 6.14 ± 0.32 cream coloured long roots having 4.40 ± 0.10 cm length and 0.62 ± 0.02 cm diameter from
Table 3 . Effect of plant growth regulators Schenk and Hildebrandt medium on tuberous root production in
NAA | IBA | IAA | 2,4-D | Number of tuberous roots | Length of tuberous roots (cm) | Diameter of tuberous roots | Color of tuberous roots |
---|---|---|---|---|---|---|---|
0.05 | - | - | - | 2.12 ± 0.12e | 2.29 ± 0.18c | 0.13 ± 0.03f | Cream |
0.1 | - | - | - | 3.70 ± 0.15d | 3.28 ± 0.19b | 0.34 ± 0.04d | Cream |
0.5 | - | - | - | 6.14 ± 0.32a | 4.40 ± 0.10a | 0.62 ± 0.12a | Cream |
1.0 | - | - | - | 4.52 ± 0.24c | 3.50 ± 0.34b | 0.53 ± 0.13b | Cream |
1.5 | - | - | - | 3.52 ± 0.35d | 3.31 ± 0.22b | 0.42 ± 0.08c | Cream |
2.0 | - | - | - | 3.23 ± 0.23d | 3.00 ± 0.26b | 0.22 ± 0.12e | White |
- | 0.05 | - | - | 1.21 ± 0.12f | 0.66 ± 0.15e | 0.55 ± 0.17b | White |
- | 0.1 | - | - | 1.03 ± 0.13f | 1.57 ± 0.29d | 0.15 ± 0.34f | White |
- | 0.5 | - | - | 5.17 ± 0.41b | 4.20 ± 0.10a | 0.54 ± 0.21b | Cream |
- | 1.0 | - | - | 4.36 ± 0.43c | 3.42 ± 0.43b | 0.33 ± 0.13d | Cream |
- | 1.5 | - | - | 4.21 ± 0.21c | 3.31 ± 0.22b | 0.42 ± 0.12c | Cream |
- | 2.0 | - | - | 3.33 ± 0.22d | 2.83 ± 0.44c | 0.33 ± 0.25d | Cream |
- | - | 0.05 | - | 1.34 ± 0.33f | 1.02 ± 0.32d | 0.53 ± 0.21b | White |
- | - | 0.1 | - | 1.44 ± 0.22f | 1.14 ± 0.11d | 0.44 ± 0.11c | White |
- | - | 0.5 | - | 1.45 ± 0.41f | 1.17 ± 0.21d | 0.34 ± 0.13d | White |
- | - | 1.0 | - | 1.64 ± 0.32f | 1.15 ± 0.30d | 0.24 ± 0.22e | White |
- | - | 1.5 | - | 1.75 ± 0.43f | 1.06 ± 0.32d | 0.21 ± 0.41e | White |
- | - | - | 0.05 | 1.13 ± 0.32f | 0.55 ± 0.43e | 0.10 ± 0.30f | White |
- | - | - | 0.1 | 1.15 ± 0.14f | 0.56 ± 0.24e | 0.15 ± 0.05f | White |
- | - | - | 0.5 | 1.16 ± 0.32f | 0.44 ± 0.33e | 0.18 ± 0.21f | White |
- | - | - | 1.0 | 1.15 ± 0.17f | 0.43 ± 0.36e | 0.15 ± 0.11f | White |
- | - | - | 1.5 | 1.14 ± 0.42f | 0.32 ± 0.21e | 0.12 ± 0.01f | White |
0.1 | 0.1 | - | - | 2.12 ± 0.37e | 1.44 ± 0.25d | 0.43 ± 0.41c | White |
0.5 | 0.1 | - | - | 3.36 ± 0.23d | 1.90 ± 0.04d | 0.32 ± 0.32d | White |
1 | 0.1 | - | - | 3.75 ± 0.26d | 1.80 ± 0.06d | 0.41 ± 0.31c | Cream |
0.5 | 0.5 | - | - | 4.14 ± 0.19c | 2.36 ± 0.21c | 0.13 ± 0.23f | Cream |
0.5 | 1 | - | - | 3.46 ± 0.27f | 2.21 ± 0.17c | 0.10 ± 0.24f | Cream |
1 | 0.5 | - | - | 4.27 ± 0.43c | 2.53 ± 0.15c | 0.12 ± 0.32f | Cream |
1 | 1 | - | - | 3.66 ± 0.39d | 1.93 ± 0.13d | 0.33 ± 0.21d | Cream |
2 | 0.5 | - | - | 2.16 ± 0.32e | 1.42 ± 0.16d | 0.24 ± 0.23e | Cream |
2 | 1.0 | - | - | 1.44 ± 0.13f | 1.36 ± 0.14d | 0.42 ± 0.12c | Cream |
NAA - α-naphthaleneacetic acid; IBA - indole 3-butyric acid; IAA - indole 3-acetic acid; 2,4-D - 2,4-dichlorophenoxyacetic acid
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05).
Leaf explants of
Table 4 . Effect of plant growth regulators in Schenk and Hildebrandt medium on tuberous root production in
NAA | IBA | Mean number of tuberous roots | Length of tuberous roots (cm) | Diameter of tubers | Color of tuberous roots |
---|---|---|---|---|---|
0.05 | - | 2.04 ± 0.10c | 2.26 ± 0.01a | 1.24 ± 0.02b | Cream |
0.1 | - | 2.76 ± 0.15b | 1.18 ± 0.01c | 1.05 ± 0.01c | Cream |
0.5 | - | 3.21 ± 0.26a | 2.00 ± 0.10b | 1.45 ± 0.03a | Cream |
1.0 | - | - | - | - | - |
2.0 | - | - | - | - | - |
3.0 | - | 1.23 ± 0.05d | 0.52 ± 0.10d | 0.16 ± 0.05d | White |
- | 0.05 | - | - | - | - |
- | 0.1 | - | - | - | - |
- | 0.5 | 1.02 ± 0.08e | 0.23 ± 0.15e | 0.12 ± 0.07e | White |
NAA - α-naphthaleneacetic acid; IBA - indole 3-butyric acid
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05).
Adventitious roots induced when subcultured to liquid SH medium containing 0.5 mg/l NAA that induced better formation of
In the present study the phytochemical analysis of methanolic extracts of
Table 5 . Phytochemical analysis of methanolic root tuber extracts of
Phytochemicals | Test | Inference of phytoconstituents | ||
---|---|---|---|---|
Alkaloids | Wagner’s test | Formation of yellow or brown precipitate | +++ | ++ |
Dragendorff’s test | Formation of a reddish-brown precipitate | ++ | ++ | |
Flavonoids | Alkaline test | A yellow color observed at the ammonia layer | + | ++ |
Shinoda test | A pink, scarlet, crimson red, or occasionally green to blue color appeared after a few minutes | +++ | + | |
Triterpenoids | Salkowski test | A red-brown color formed at the interface | ++ | + |
Tannins | FeCl3 test | Formation of bluish black color | + | + |
Phenols | - | Formation of greenish-black color | + | + |
Anthraquinones | - | Appearance of red color | - | - |
Carbohydrates | Molisch’s test | A reddish-violet or purple ring at the junction of the two liquids | ++ | + |
Saponins | Foam test | Stable foam | − | − |
Protein | Biuret test | Formation of purple or violet color | + | + |
Phytosterol | Liebermann-Burchard test | A brown ring formation at the junction | ++ | − |
(‘+’ for presence, ‘−’ for absence)
Lupeol is an immense bioactive triterpenoid present in different medicinal plants having anti-inflammatory, anti-microbial, anti-protozoal, anti-tumor, anti-proliferative, anti-invasive, and cholesterol lowering properties. Chromatographic separation of methanolic extracts of
HPTLC is a valuable quality assessment tool for the evaluation of botanical samples efficiently and offers high degree of selectivity, sensitivity and rapidity along with single-step sample preparation. Also it is a reliable method for the quantification of nanograms level of samples. This technique was explored by Sunil et al. (2017) who reported the antioxidant activity of a bioassay-guided fractionation and its active components/compounds in
The present study standardized a novel consistent system of
PTS and ASH conceptualized the study, SPS executed the research work, PTS and ASH analysed the data and written the manuscript.
Authors have no conflict of interest
Authors declare that this manuscript has not been submitted fully or partially anywhere.
We are thankful to the Director, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram 695562, Kerala, India for providing the facility for HPTLC analysis of the samples.
J Plant Biotechnol 2022; 49(3): 230-239
Published online September 30, 2022 https://doi.org/10.5010/JPB.2022.49.3.230
Copyright © The Korean Society of Plant Biotechnology.
Padmavathi Amma Somasekharan Nair Smitha Devi ・Achuthan Sudarsanan Hemanthakumar ・Thankappan Suvarna Preetha
Plant Tissue Culture Laboratory, Department of Botany, University College, Thiruvananthapuram 695034, Kerala, India; Research Centre, University of Kerala, Kerala, India
Biotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram 695562, Kerala, India
Correspondence to:e-mail: preethahemanth@yahoo.com
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Holostemma annulare (Family Asclepiadaceae) is an invaluable vulnerable medicinal plant; the root tubers are used in Ayurveda medicine and by folk healers to treat various ailments. In this study, Schenk and Hildebrandt medium fortified with the cytokinins 6-benzyl adenine, kinetin, and auxins, including indole 3-butyric acid, indole 3-acetic acid, α-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid, were checked for their efficiency on root tuber induction from different explants. Adventitious root tubers were more successfully induced from in vitro leaf segments and shoots when cultured in Schenk and Hildebrandt medium supplemented with 0.5 mg/l of α-naphthaleneacetic acid. In addition, preliminary phytochemical analysis of in vitro root tubers and identification of different secondary metabolites were conducted. Thin layer chromatography and high performance thin layer chromatography analysis of the crude methanolic extracts of the in vitro root tuber identified the presence of lupeol, a bioactive triterpene. Adventitious root tuber induction offers a novel method for the in vitro production of bioactive metabolites that can be scaled up by bioreactors, thus ensuring the conservation and sustainable utilization of H. annulare. The study warrants further scale-up production and pharmacological investigation that can be extended for pharmaceutical needs.
Keywords: Adventitious in vitro root tubers, Holostemma annulare, high performance thin layer chromatography (HPTLC), lupeol, Schenk and Hildebrandt (SH) medium
The tuberous roots of
Explants like nodes and shoot tips collected from disease-free plants of
The
Adventitious roots induced were subcultured to liquid SH medium containing the growth regulator that evoked better induction of
For detecting the presence of bioactive metabolites in the
Chromatographic separation of bioactive component
About 2.25 g
HPTLC analysis of methanolic extracts of root samples (used for TLC separation) were carried out in HPTLC system (CAMAG, Switzerland), CAMAG twin-trough plate development chamber, using silica gel plates (60F254 Manufacturer E. MERCK KGaA). Sample application was carried out on CAMAG Linomat 5 instrument (CAMAG TLC Scanner 3, CAMAG Reprostar 3 photodocument system and WinCATS Software 4.03). The submitted samples are applied to a Silica gel 60 F254 TLC plate (E. Merck, Germany) (10 cm × 10 cm) using Linomat V sample applicator as 8 mm wide bands along with the standard lupeol (1 mg lupeol dissolved in 1 ml chloroform). It is then developed up to 80 mm in a twin trough glass chamber using the Mobile Phase: 16:4, hexane : ethyl acetate. The plate is then derivatised using anisaldehyde sulphuric acid reagent, heated at 110°C for 10 min. The plate is then scanned densitometrically at 580 nm using TLC Scanner 3 equipped with WinCats software. The derivatised plate is then photo documented using CAMAG reprostar 3.
Each
When the effect of different medium formulations such as MS, ½ MS and SH media were tested for shoot culture initiation in
Table 1 . Effect of plant growth regulators in Schenk and Hildebrandt medium on shoot culture initiation in
BA | Kinetin | NAA | Number of shoots | Length of shoots (cm) | Number of nodes per shoots | |||
---|---|---|---|---|---|---|---|---|
Node | Shoot tip | Node | Shoot tip | Node | Shoot tip | |||
0.25 | - | - | 1.88 ± 0.01b | 1.83 ± 0.44b | 11.91 ± 0.01b | 10.21 ± 0.21b | 5.51 ± 0.31b | 5.83 ± 0.10b |
0.5 | - | - | 1.95 ± 0.12a | 1.92 ± 0.21a | 10.82 ± 0.02c | 10.01 ± 0.43b | 6.31 ± 0.31a | 6.16 ± 0.05a |
1.0 | - | - | 1.45 ± 0.37d | 1.56 ± 0.42e | 7.38 ± 0.01f | 8.21 ± 0.31d | 5.91 ± 0.43b | 5.71 ± 0.03b |
2.0 | - | - | 1.34 ± 0.36e | 1.36 ± 0.16f | 6.21 ± 0.01g | 6.41 ± 0.36f | 5.12 ± 0.22b | 5.13 ± 0.11b |
0.25 | 0.5 | - | 1.67 ± 0.43c | 1.66 ± 0.21d | 10.52 ± 0.02c | 9.22 ± 0.41c | 3.92 ± 0.32e | 2.63 ± 0.22e |
0.5 | 0.5 | - | 1.24 ± 0.32f | 1.77 ± 0.43c | 8.23 ± 0.03e | 8.03 ± 0.26d | 3.22 ± 0.23e | 3.34 ± 0.04d |
1.0 | 0.5 | - | 1.13 ± 0.04g | 1.15 ± 0.09g | 6.24 ± 0.04g | 6.14 ± 0.13f | 3.22 ± 0.22e | 3.23 ± 0.13d |
2.0 | 0.5 | - | 1.04 ± 0.08h | 1.05 ± 0.05h | 4.03 ± 0.02h | 3.92 ± 0.02g | 2.82 ± 0.41f | 2.74 ± 0.33e |
0.25 | - | 0.1 | 1.97 ± 0.21a | 1.95 ± 0.03a | 13.72 ± 0.02a | 12.83 ± 0.13a | 6.52 ± 0.28a | 6.43 ± 0.42a |
0.5 | - | 0.5 | 1.85 ± 0.42b | 1.73 ± 0.07c | 9.52 ± 0.02d | 9.33 ± 0.23c | 4.34 ± 0.34d | 4.23 ± 0.17c |
1.0 | - | 1.0 | 1.64 ± 0.11c | 1.54 ± 0.20e | 8.43 ± 0.03e | 8.23 ± 0.25d | 4.12 ± 0.13d | 4.33 ± 0.21c |
2.0 | - | 1.5 | 1.22 ± 0.32f | 1.34 ± 0.72f | 7.93 ± 0.03f | 7.53 ± 0.28e | 3.22 ± 0.15e | 3.32 ± 0.32d |
BA - 6-benzyl adenine; NAA - α-naphthaleneacetic acid.
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05)..
The initiated shoots subcultured to SH medium supplemented with 2.0 mg/l BA in combination with 0.5 mg/l Kinetin and 1.0 mg/l IBA evoked maximum 7.16 ± 0.15 shoots with a mean length of 6.53 ± 0.03 cm and 4.53 ± 0.05 nodes per shoot (Fig. 1d) (Table 2). Likewise, Gayathri et al. (2009) reported multiple shoot induction in
Table 2 . Effect of plant growth regulators in Schenk and Hildebrandt medium on shoot multiplication in
BA | Kinetin | IBA | NAA | Number of shoots | Length of shoots (cm) | Number of nodes per shoots |
---|---|---|---|---|---|---|
1.0 | 0.5 | 2.13 ± 0.15f | 2.21 ± 0.31e | 2.06 ± 0.05c | ||
2.0 | 0.5 | - | - | 2.23 ± 0.25f | 2.11 ± 0.20e | 1.62 ± 0.41d |
2.0 | 1.0 | - | - | 3.14 ± 0.10e | 2.52 ± 0.24e | 2.23 ± 0.10c |
1.0 | - | 0.5 | 4.23 ± 0.20d | 3.21 ± 0.01d | 2.63 ± 0.25c | |
2.0 | - | 0.5 | - | 4.06 ± 0.20d | 2.42 ± 0.02e | 3.13 ± 0.11b |
2.0 | - | 1.0 | - | 3.36 ± 0.35e | 2.82 ± 0.02e | 3.43 ± 0.20b |
1.0 | - | - | 0.5 | 4.26 ± 0.20d | 1.23 ± 0.03f | 2.41 ± 0.36c |
2.0 | - | - | 0.5 | 3.33 ± 0.28e | 1.81 ± 0.10f | 2.44 ± 0.05c |
2.0 | - | - | 1.0 | 3.1 ± 0.100e | 2.07 ± 0.06e | 2.32 ± 0.10c |
1.0 | 0.5 | 0.5 | - | 4.06 ± 0.05d | 5.23 ± 0.03b | 3.43 ± 0.32b |
2.0 | 0.5 | 0.5 | - | 6.13 ± 0.15b | 6.33 ± 0.25a | 4.46 ± 0.28a |
2.0 | 0.5 | 1.0 | - | 7.16 ± 0.15a | 6.53 ± 0.03a | 4.53 ± 0.05a |
1.0 | 0.5 | - | 0.5 | 4.06 ± 0.05d | 4.23 ± 0.15c | 3.12 ± 0.10b |
2.0 | 0.5 | - | 1.0 | 5.16 ± 0.15c | 5.33 ± 0.13b | 3.16 ± 0.20b |
2.0 | 1.0 | - | 1.0 | 3.13 ± 0.12e | 4.19 ± 0.06c | 2.33 ± 0.30c |
BA - 6-benzyl adenine; IBA - indole 3-butyric acid; NAA - α-naphthaleneacetic acid.
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05)..
Better root tuber induction devoid of callusing was noticed in SH medium containing 0.5 mg/l NAA (Fig. 1e-g) that produced 6.14 ± 0.32 cream coloured long roots having 4.40 ± 0.10 cm length and 0.62 ± 0.02 cm diameter from
Table 3 . Effect of plant growth regulators Schenk and Hildebrandt medium on tuberous root production in
NAA | IBA | IAA | 2,4-D | Number of tuberous roots | Length of tuberous roots (cm) | Diameter of tuberous roots | Color of tuberous roots |
---|---|---|---|---|---|---|---|
0.05 | - | - | - | 2.12 ± 0.12e | 2.29 ± 0.18c | 0.13 ± 0.03f | Cream |
0.1 | - | - | - | 3.70 ± 0.15d | 3.28 ± 0.19b | 0.34 ± 0.04d | Cream |
0.5 | - | - | - | 6.14 ± 0.32a | 4.40 ± 0.10a | 0.62 ± 0.12a | Cream |
1.0 | - | - | - | 4.52 ± 0.24c | 3.50 ± 0.34b | 0.53 ± 0.13b | Cream |
1.5 | - | - | - | 3.52 ± 0.35d | 3.31 ± 0.22b | 0.42 ± 0.08c | Cream |
2.0 | - | - | - | 3.23 ± 0.23d | 3.00 ± 0.26b | 0.22 ± 0.12e | White |
- | 0.05 | - | - | 1.21 ± 0.12f | 0.66 ± 0.15e | 0.55 ± 0.17b | White |
- | 0.1 | - | - | 1.03 ± 0.13f | 1.57 ± 0.29d | 0.15 ± 0.34f | White |
- | 0.5 | - | - | 5.17 ± 0.41b | 4.20 ± 0.10a | 0.54 ± 0.21b | Cream |
- | 1.0 | - | - | 4.36 ± 0.43c | 3.42 ± 0.43b | 0.33 ± 0.13d | Cream |
- | 1.5 | - | - | 4.21 ± 0.21c | 3.31 ± 0.22b | 0.42 ± 0.12c | Cream |
- | 2.0 | - | - | 3.33 ± 0.22d | 2.83 ± 0.44c | 0.33 ± 0.25d | Cream |
- | - | 0.05 | - | 1.34 ± 0.33f | 1.02 ± 0.32d | 0.53 ± 0.21b | White |
- | - | 0.1 | - | 1.44 ± 0.22f | 1.14 ± 0.11d | 0.44 ± 0.11c | White |
- | - | 0.5 | - | 1.45 ± 0.41f | 1.17 ± 0.21d | 0.34 ± 0.13d | White |
- | - | 1.0 | - | 1.64 ± 0.32f | 1.15 ± 0.30d | 0.24 ± 0.22e | White |
- | - | 1.5 | - | 1.75 ± 0.43f | 1.06 ± 0.32d | 0.21 ± 0.41e | White |
- | - | - | 0.05 | 1.13 ± 0.32f | 0.55 ± 0.43e | 0.10 ± 0.30f | White |
- | - | - | 0.1 | 1.15 ± 0.14f | 0.56 ± 0.24e | 0.15 ± 0.05f | White |
- | - | - | 0.5 | 1.16 ± 0.32f | 0.44 ± 0.33e | 0.18 ± 0.21f | White |
- | - | - | 1.0 | 1.15 ± 0.17f | 0.43 ± 0.36e | 0.15 ± 0.11f | White |
- | - | - | 1.5 | 1.14 ± 0.42f | 0.32 ± 0.21e | 0.12 ± 0.01f | White |
0.1 | 0.1 | - | - | 2.12 ± 0.37e | 1.44 ± 0.25d | 0.43 ± 0.41c | White |
0.5 | 0.1 | - | - | 3.36 ± 0.23d | 1.90 ± 0.04d | 0.32 ± 0.32d | White |
1 | 0.1 | - | - | 3.75 ± 0.26d | 1.80 ± 0.06d | 0.41 ± 0.31c | Cream |
0.5 | 0.5 | - | - | 4.14 ± 0.19c | 2.36 ± 0.21c | 0.13 ± 0.23f | Cream |
0.5 | 1 | - | - | 3.46 ± 0.27f | 2.21 ± 0.17c | 0.10 ± 0.24f | Cream |
1 | 0.5 | - | - | 4.27 ± 0.43c | 2.53 ± 0.15c | 0.12 ± 0.32f | Cream |
1 | 1 | - | - | 3.66 ± 0.39d | 1.93 ± 0.13d | 0.33 ± 0.21d | Cream |
2 | 0.5 | - | - | 2.16 ± 0.32e | 1.42 ± 0.16d | 0.24 ± 0.23e | Cream |
2 | 1.0 | - | - | 1.44 ± 0.13f | 1.36 ± 0.14d | 0.42 ± 0.12c | Cream |
NAA - α-naphthaleneacetic acid; IBA - indole 3-butyric acid; IAA - indole 3-acetic acid; 2,4-D - 2,4-dichlorophenoxyacetic acid.
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05)..
Leaf explants of
Table 4 . Effect of plant growth regulators in Schenk and Hildebrandt medium on tuberous root production in
NAA | IBA | Mean number of tuberous roots | Length of tuberous roots (cm) | Diameter of tubers | Color of tuberous roots |
---|---|---|---|---|---|
0.05 | - | 2.04 ± 0.10c | 2.26 ± 0.01a | 1.24 ± 0.02b | Cream |
0.1 | - | 2.76 ± 0.15b | 1.18 ± 0.01c | 1.05 ± 0.01c | Cream |
0.5 | - | 3.21 ± 0.26a | 2.00 ± 0.10b | 1.45 ± 0.03a | Cream |
1.0 | - | - | - | - | - |
2.0 | - | - | - | - | - |
3.0 | - | 1.23 ± 0.05d | 0.52 ± 0.10d | 0.16 ± 0.05d | White |
- | 0.05 | - | - | - | - |
- | 0.1 | - | - | - | - |
- | 0.5 | 1.02 ± 0.08e | 0.23 ± 0.15e | 0.12 ± 0.07e | White |
NAA - α-naphthaleneacetic acid; IBA - indole 3-butyric acid.
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05)..
Adventitious roots induced when subcultured to liquid SH medium containing 0.5 mg/l NAA that induced better formation of
In the present study the phytochemical analysis of methanolic extracts of
Table 5 . Phytochemical analysis of methanolic root tuber extracts of
Phytochemicals | Test | Inference of phytoconstituents | ||
---|---|---|---|---|
Alkaloids | Wagner’s test | Formation of yellow or brown precipitate | +++ | ++ |
Dragendorff’s test | Formation of a reddish-brown precipitate | ++ | ++ | |
Flavonoids | Alkaline test | A yellow color observed at the ammonia layer | + | ++ |
Shinoda test | A pink, scarlet, crimson red, or occasionally green to blue color appeared after a few minutes | +++ | + | |
Triterpenoids | Salkowski test | A red-brown color formed at the interface | ++ | + |
Tannins | FeCl3 test | Formation of bluish black color | + | + |
Phenols | - | Formation of greenish-black color | + | + |
Anthraquinones | - | Appearance of red color | - | - |
Carbohydrates | Molisch’s test | A reddish-violet or purple ring at the junction of the two liquids | ++ | + |
Saponins | Foam test | Stable foam | − | − |
Protein | Biuret test | Formation of purple or violet color | + | + |
Phytosterol | Liebermann-Burchard test | A brown ring formation at the junction | ++ | − |
(‘+’ for presence, ‘−’ for absence).
Lupeol is an immense bioactive triterpenoid present in different medicinal plants having anti-inflammatory, anti-microbial, anti-protozoal, anti-tumor, anti-proliferative, anti-invasive, and cholesterol lowering properties. Chromatographic separation of methanolic extracts of
HPTLC is a valuable quality assessment tool for the evaluation of botanical samples efficiently and offers high degree of selectivity, sensitivity and rapidity along with single-step sample preparation. Also it is a reliable method for the quantification of nanograms level of samples. This technique was explored by Sunil et al. (2017) who reported the antioxidant activity of a bioassay-guided fractionation and its active components/compounds in
The present study standardized a novel consistent system of
PTS and ASH conceptualized the study, SPS executed the research work, PTS and ASH analysed the data and written the manuscript.
Authors have no conflict of interest
Authors declare that this manuscript has not been submitted fully or partially anywhere.
We are thankful to the Director, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram 695562, Kerala, India for providing the facility for HPTLC analysis of the samples.
Table 1 . Effect of plant growth regulators in Schenk and Hildebrandt medium on shoot culture initiation in
BA | Kinetin | NAA | Number of shoots | Length of shoots (cm) | Number of nodes per shoots | |||
---|---|---|---|---|---|---|---|---|
Node | Shoot tip | Node | Shoot tip | Node | Shoot tip | |||
0.25 | - | - | 1.88 ± 0.01b | 1.83 ± 0.44b | 11.91 ± 0.01b | 10.21 ± 0.21b | 5.51 ± 0.31b | 5.83 ± 0.10b |
0.5 | - | - | 1.95 ± 0.12a | 1.92 ± 0.21a | 10.82 ± 0.02c | 10.01 ± 0.43b | 6.31 ± 0.31a | 6.16 ± 0.05a |
1.0 | - | - | 1.45 ± 0.37d | 1.56 ± 0.42e | 7.38 ± 0.01f | 8.21 ± 0.31d | 5.91 ± 0.43b | 5.71 ± 0.03b |
2.0 | - | - | 1.34 ± 0.36e | 1.36 ± 0.16f | 6.21 ± 0.01g | 6.41 ± 0.36f | 5.12 ± 0.22b | 5.13 ± 0.11b |
0.25 | 0.5 | - | 1.67 ± 0.43c | 1.66 ± 0.21d | 10.52 ± 0.02c | 9.22 ± 0.41c | 3.92 ± 0.32e | 2.63 ± 0.22e |
0.5 | 0.5 | - | 1.24 ± 0.32f | 1.77 ± 0.43c | 8.23 ± 0.03e | 8.03 ± 0.26d | 3.22 ± 0.23e | 3.34 ± 0.04d |
1.0 | 0.5 | - | 1.13 ± 0.04g | 1.15 ± 0.09g | 6.24 ± 0.04g | 6.14 ± 0.13f | 3.22 ± 0.22e | 3.23 ± 0.13d |
2.0 | 0.5 | - | 1.04 ± 0.08h | 1.05 ± 0.05h | 4.03 ± 0.02h | 3.92 ± 0.02g | 2.82 ± 0.41f | 2.74 ± 0.33e |
0.25 | - | 0.1 | 1.97 ± 0.21a | 1.95 ± 0.03a | 13.72 ± 0.02a | 12.83 ± 0.13a | 6.52 ± 0.28a | 6.43 ± 0.42a |
0.5 | - | 0.5 | 1.85 ± 0.42b | 1.73 ± 0.07c | 9.52 ± 0.02d | 9.33 ± 0.23c | 4.34 ± 0.34d | 4.23 ± 0.17c |
1.0 | - | 1.0 | 1.64 ± 0.11c | 1.54 ± 0.20e | 8.43 ± 0.03e | 8.23 ± 0.25d | 4.12 ± 0.13d | 4.33 ± 0.21c |
2.0 | - | 1.5 | 1.22 ± 0.32f | 1.34 ± 0.72f | 7.93 ± 0.03f | 7.53 ± 0.28e | 3.22 ± 0.15e | 3.32 ± 0.32d |
BA - 6-benzyl adenine; NAA - α-naphthaleneacetic acid.
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05)..
Table 2 . Effect of plant growth regulators in Schenk and Hildebrandt medium on shoot multiplication in
BA | Kinetin | IBA | NAA | Number of shoots | Length of shoots (cm) | Number of nodes per shoots |
---|---|---|---|---|---|---|
1.0 | 0.5 | 2.13 ± 0.15f | 2.21 ± 0.31e | 2.06 ± 0.05c | ||
2.0 | 0.5 | - | - | 2.23 ± 0.25f | 2.11 ± 0.20e | 1.62 ± 0.41d |
2.0 | 1.0 | - | - | 3.14 ± 0.10e | 2.52 ± 0.24e | 2.23 ± 0.10c |
1.0 | - | 0.5 | 4.23 ± 0.20d | 3.21 ± 0.01d | 2.63 ± 0.25c | |
2.0 | - | 0.5 | - | 4.06 ± 0.20d | 2.42 ± 0.02e | 3.13 ± 0.11b |
2.0 | - | 1.0 | - | 3.36 ± 0.35e | 2.82 ± 0.02e | 3.43 ± 0.20b |
1.0 | - | - | 0.5 | 4.26 ± 0.20d | 1.23 ± 0.03f | 2.41 ± 0.36c |
2.0 | - | - | 0.5 | 3.33 ± 0.28e | 1.81 ± 0.10f | 2.44 ± 0.05c |
2.0 | - | - | 1.0 | 3.1 ± 0.100e | 2.07 ± 0.06e | 2.32 ± 0.10c |
1.0 | 0.5 | 0.5 | - | 4.06 ± 0.05d | 5.23 ± 0.03b | 3.43 ± 0.32b |
2.0 | 0.5 | 0.5 | - | 6.13 ± 0.15b | 6.33 ± 0.25a | 4.46 ± 0.28a |
2.0 | 0.5 | 1.0 | - | 7.16 ± 0.15a | 6.53 ± 0.03a | 4.53 ± 0.05a |
1.0 | 0.5 | - | 0.5 | 4.06 ± 0.05d | 4.23 ± 0.15c | 3.12 ± 0.10b |
2.0 | 0.5 | - | 1.0 | 5.16 ± 0.15c | 5.33 ± 0.13b | 3.16 ± 0.20b |
2.0 | 1.0 | - | 1.0 | 3.13 ± 0.12e | 4.19 ± 0.06c | 2.33 ± 0.30c |
BA - 6-benzyl adenine; IBA - indole 3-butyric acid; NAA - α-naphthaleneacetic acid.
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05)..
Table 3 . Effect of plant growth regulators Schenk and Hildebrandt medium on tuberous root production in
NAA | IBA | IAA | 2,4-D | Number of tuberous roots | Length of tuberous roots (cm) | Diameter of tuberous roots | Color of tuberous roots |
---|---|---|---|---|---|---|---|
0.05 | - | - | - | 2.12 ± 0.12e | 2.29 ± 0.18c | 0.13 ± 0.03f | Cream |
0.1 | - | - | - | 3.70 ± 0.15d | 3.28 ± 0.19b | 0.34 ± 0.04d | Cream |
0.5 | - | - | - | 6.14 ± 0.32a | 4.40 ± 0.10a | 0.62 ± 0.12a | Cream |
1.0 | - | - | - | 4.52 ± 0.24c | 3.50 ± 0.34b | 0.53 ± 0.13b | Cream |
1.5 | - | - | - | 3.52 ± 0.35d | 3.31 ± 0.22b | 0.42 ± 0.08c | Cream |
2.0 | - | - | - | 3.23 ± 0.23d | 3.00 ± 0.26b | 0.22 ± 0.12e | White |
- | 0.05 | - | - | 1.21 ± 0.12f | 0.66 ± 0.15e | 0.55 ± 0.17b | White |
- | 0.1 | - | - | 1.03 ± 0.13f | 1.57 ± 0.29d | 0.15 ± 0.34f | White |
- | 0.5 | - | - | 5.17 ± 0.41b | 4.20 ± 0.10a | 0.54 ± 0.21b | Cream |
- | 1.0 | - | - | 4.36 ± 0.43c | 3.42 ± 0.43b | 0.33 ± 0.13d | Cream |
- | 1.5 | - | - | 4.21 ± 0.21c | 3.31 ± 0.22b | 0.42 ± 0.12c | Cream |
- | 2.0 | - | - | 3.33 ± 0.22d | 2.83 ± 0.44c | 0.33 ± 0.25d | Cream |
- | - | 0.05 | - | 1.34 ± 0.33f | 1.02 ± 0.32d | 0.53 ± 0.21b | White |
- | - | 0.1 | - | 1.44 ± 0.22f | 1.14 ± 0.11d | 0.44 ± 0.11c | White |
- | - | 0.5 | - | 1.45 ± 0.41f | 1.17 ± 0.21d | 0.34 ± 0.13d | White |
- | - | 1.0 | - | 1.64 ± 0.32f | 1.15 ± 0.30d | 0.24 ± 0.22e | White |
- | - | 1.5 | - | 1.75 ± 0.43f | 1.06 ± 0.32d | 0.21 ± 0.41e | White |
- | - | - | 0.05 | 1.13 ± 0.32f | 0.55 ± 0.43e | 0.10 ± 0.30f | White |
- | - | - | 0.1 | 1.15 ± 0.14f | 0.56 ± 0.24e | 0.15 ± 0.05f | White |
- | - | - | 0.5 | 1.16 ± 0.32f | 0.44 ± 0.33e | 0.18 ± 0.21f | White |
- | - | - | 1.0 | 1.15 ± 0.17f | 0.43 ± 0.36e | 0.15 ± 0.11f | White |
- | - | - | 1.5 | 1.14 ± 0.42f | 0.32 ± 0.21e | 0.12 ± 0.01f | White |
0.1 | 0.1 | - | - | 2.12 ± 0.37e | 1.44 ± 0.25d | 0.43 ± 0.41c | White |
0.5 | 0.1 | - | - | 3.36 ± 0.23d | 1.90 ± 0.04d | 0.32 ± 0.32d | White |
1 | 0.1 | - | - | 3.75 ± 0.26d | 1.80 ± 0.06d | 0.41 ± 0.31c | Cream |
0.5 | 0.5 | - | - | 4.14 ± 0.19c | 2.36 ± 0.21c | 0.13 ± 0.23f | Cream |
0.5 | 1 | - | - | 3.46 ± 0.27f | 2.21 ± 0.17c | 0.10 ± 0.24f | Cream |
1 | 0.5 | - | - | 4.27 ± 0.43c | 2.53 ± 0.15c | 0.12 ± 0.32f | Cream |
1 | 1 | - | - | 3.66 ± 0.39d | 1.93 ± 0.13d | 0.33 ± 0.21d | Cream |
2 | 0.5 | - | - | 2.16 ± 0.32e | 1.42 ± 0.16d | 0.24 ± 0.23e | Cream |
2 | 1.0 | - | - | 1.44 ± 0.13f | 1.36 ± 0.14d | 0.42 ± 0.12c | Cream |
NAA - α-naphthaleneacetic acid; IBA - indole 3-butyric acid; IAA - indole 3-acetic acid; 2,4-D - 2,4-dichlorophenoxyacetic acid.
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05)..
Table 4 . Effect of plant growth regulators in Schenk and Hildebrandt medium on tuberous root production in
NAA | IBA | Mean number of tuberous roots | Length of tuberous roots (cm) | Diameter of tubers | Color of tuberous roots |
---|---|---|---|---|---|
0.05 | - | 2.04 ± 0.10c | 2.26 ± 0.01a | 1.24 ± 0.02b | Cream |
0.1 | - | 2.76 ± 0.15b | 1.18 ± 0.01c | 1.05 ± 0.01c | Cream |
0.5 | - | 3.21 ± 0.26a | 2.00 ± 0.10b | 1.45 ± 0.03a | Cream |
1.0 | - | - | - | - | - |
2.0 | - | - | - | - | - |
3.0 | - | 1.23 ± 0.05d | 0.52 ± 0.10d | 0.16 ± 0.05d | White |
- | 0.05 | - | - | - | - |
- | 0.1 | - | - | - | - |
- | 0.5 | 1.02 ± 0.08e | 0.23 ± 0.15e | 0.12 ± 0.07e | White |
NAA - α-naphthaleneacetic acid; IBA - indole 3-butyric acid.
Data represents the mean ± SE of ten replicates, repeated thrice. Mean values followed by the same superscript letter do not differ significantly based on the ANOVA and Duncan’s multiple range test (p ≤ 0.05)..
Table 5 . Phytochemical analysis of methanolic root tuber extracts of
Phytochemicals | Test | Inference of phytoconstituents | ||
---|---|---|---|---|
Alkaloids | Wagner’s test | Formation of yellow or brown precipitate | +++ | ++ |
Dragendorff’s test | Formation of a reddish-brown precipitate | ++ | ++ | |
Flavonoids | Alkaline test | A yellow color observed at the ammonia layer | + | ++ |
Shinoda test | A pink, scarlet, crimson red, or occasionally green to blue color appeared after a few minutes | +++ | + | |
Triterpenoids | Salkowski test | A red-brown color formed at the interface | ++ | + |
Tannins | FeCl3 test | Formation of bluish black color | + | + |
Phenols | - | Formation of greenish-black color | + | + |
Anthraquinones | - | Appearance of red color | - | - |
Carbohydrates | Molisch’s test | A reddish-violet or purple ring at the junction of the two liquids | ++ | + |
Saponins | Foam test | Stable foam | − | − |
Protein | Biuret test | Formation of purple or violet color | + | + |
Phytosterol | Liebermann-Burchard test | A brown ring formation at the junction | ++ | − |
(‘+’ for presence, ‘−’ for absence).
Journal of
Plant Biotechnology