The ever-rising demand for natural products and continuously diminishing plant population in natural habitat necessitates to develop alternative method to generate herbal ingredients of industrial importance. Hence, in vitro adventitious roots of V. jatamansi are worked out in this study as a substitute to obtain valerenic acid derivatives, essential oil and other derivatives.
Induction of adventitious root cultures
Murashige and Skoog (1962) Schenk and Hildebrandt (1972) and Gamborg (1968) medium were tried to induce in vitro adventitious roots from leaf explant of V. jatamansi. For this purpose, leaves were surface sterilized and cut into 4–5 mm sections and inoculated on different hormone free media under aseptic conditions.
The selection of explant was based on earlier reports that showed leaf explant as a best source for initiation of adventitious roots cultures in P. vietnamensis (Trinh et al. 2012; Tam et al. 2015; Linh et al. 2019) C. tenuiflora (Gómez-Aguirre et al. 2015) and P. multiflorum (Ho et al. 2019). Among different media tried, the induction of adventitious root was only observed in hormone free SH medium after four weeks of inoculation. This selective response of adventitious root induction might be due to variation in macronutrient, micronutrient and vitamins (myo-inositol) composition of respective basal media (SH, B5 and MS medium).
For instance, SH media have low ammonium: nitrate ratio (1:9) as compared to MS medium (1:2), which probably be helping in organogenesis. SH media also have 10 times higher myo-inositol (1000 mg/L) concertation than B5 and MS medium. It is pertinent to mention that myo-inositol known to accelerate cell division rather than increasing the cell size (Staudt 1984), which may be one of the reason for induction of adventitious root in SH medium. Similar observation was reported for adventitious root growth in five species of Scutellaria genus in SH medium fortified with IBA (1.0 mg/L) as compared to B5 and MS medium (Barska et al. 2011).
In general, adventitious root formation is a complex process and tightly regulated by various phytohormones. However, auxin is a key hormone for induction and regulation of adventitious root development. There are two types of auxins i.e. synthetic (NAA [1-naphthaleneacetic acid]) and natural analogue (IAA [indole-3-acetic acid] and IBA), which are routinely used in plant tissue culture (Bartel et al. 2001; Piotrowska-Niczyporuk et al. 2014). Therefore, the further improvement in adventitious root induction was attempted by using natural auxin analogue ‘IBA’. The basal SH medium was fortified with various concentration of IBA (0.49, 2.46, 4.92, 9.84 & 19.69 µM). The induction of adventitious roots was evident as hair-like outgrowth from cut ends of the leaves within 8 days of inoculation, as compared to 28 days in hormone free medium. Furthermore, induction of roots was also observed from the margins of the leaf sections at several points during extended period up to four weeks of incubation. Experimental data releveled a significantly (p ≤ 0.05) high percentage of adventitious root induction (90%) as well as their number (5.72 ± 0.18) and length (1.73 ± 0.06 cm) in medium fortified with 9.84 µM IBA (Table 1). However, the rooting potential found to be decreased beyond this concentration of IBA. It was noticed that above this IBA concertation i.e. 9.84 µM IBA, leaves showed more callusing than adventitious root formation. It can be deduced from the results that SH medium with 9.84 µM IBA fortification is efficient for inducing adventitious roots from V. jatamansi leaf explant at reasonably shorter time as compared to other combinations.
Table 1
Differential response of adventitious root induction with varying concentrations IBA growing on SH semi-solid medium with 3% sucrose after 4 weeks of incubation.
IBA (µM)
|
Induction %
|
No. of adventitious root (NoAR)
|
Length of adventitious root (cm) (LoAR)
|
Control
|
10.00 ± 10.00a
|
1.50 ± 0.29a
|
0.15 ± 0.03a
|
0.49
|
30.00 ± 19.14ab
|
2.33 ± 0.21b
|
0.38 ± 0.02b
|
2.46
|
40.00 ± 24.49ab
|
2.50 ± 0.19b
|
0.46 ± 0.05b
|
4.92
|
65.00 ± 23.62ab
|
3.38 ± 0.14c
|
0.70 ± 0.06c
|
9.84
|
90.00 ± 10.00b
|
5.72 ± 0.18e
|
1.73 ± 0.06e
|
19.69
|
80.00 ± 20.00b
|
4.69 ± 0.18d
|
1.16 ± 0.06d
|
Values within the column followed by different letters significantly different at p ≤ 0.05 level as determined using Duncan’s multiple range test. A value represents mean ± standard error. |
In accordance to present study, IBA has been demonstrated to be an efficient hormone for induction and multiplication of adventitious roots in number of plants (Chan et al. 2004; Agullo-Anton et al. 2011; Ghimire et al. 2018). In general, critical auxin concentration generates several signals which influence the induction as well as other phases of adventitious root development. Auxin generally promote the cell division in the initiation phase and leads to the formation of root primordia. The level of auxin in the explant is regulated by oxidation, amino acid conjugation and glycosylation (Jackson et al. 2002; Staswick 2009).
In Panax ginseng, IBA was reported to have better adventitious root induction efficiency as compared to NAA in SH medium (Kim et al. 2003). Similarly, Jayakodi et al. (2014) and Linh et al. (2019) also reported highest adventitious root induction on SH medium containing IBA from leaf explant of P. ginseng and P. vietnamensis, respectively. In case of V. jatamansi, supplementation of MS medium with BAP (6 - benzylaminopurine) and NAA (2.0 mg/L) found to promote rhizogenesis from leaves of micro propagated plants (Saini et al. 2018).
The effect of auxin on adventitious root induction was further confirmed by fortification of basal SH, MS and B5 media with optimized concentration of IBA (9.84 µM). Surprisingly, adventitious roots were induced from inoculated leaf explants in all the media. However, significantly high (p ≤ 0.05) rhizogenic potentials in terms of induction (90.0%) as well as number (5.44 ± 0.32) and length (1.47 ± 0.09 cm) of adventitious roots was obtained in SH medium as compared to MS and B5 (Fig. 1). Thus, the earlier results hold true that the SH medium with 9.84 µM IBA concentration was optimum for induction of adventitious roots. These roots were repeatedly sub-cultured on the above optimized medium for further multiplication and maintenance of mother stock. Consistent with observation of present investigation, Kim et al. (2003) described the use of SH media fortified with IBA for inducing and yielding high adventitious roots biomass in Korean ginseng as compared to NAA. However, the concentration of IBA (24.6 µM) used in this study was 2.5 times higher as compared to present work. In addition, Nhut et al. (2012) also induced adventitious roots in Vietnamese ginseng (Panax vietnamensis) on SH and MS media. Similar to our results, authors observed highest root proliferation on SH medium, however, with NAA fortification. It is hypothesized by the authors that ammonium and nitrate ratio as well vitamins (thiamine-HCL and myo-inositol) concentration of SH media might have played a significant role in high rate of adventitious root formation. Vitamin B or thiamine-HCL known to promote better root growth, thus helps in better nutrient absorption. Whereas, myo-inositol take part in the auxin (IAA) transport and play a significant role in plant cell growth and development (Loewus and Murthy 2000).
Effect of IBA on multiplication of adventitious roots in submerged cultivation
In order to evaluate the capability of adventitious roots for large-scale cultivation, in vitro induced roots were inoculated (1.0 % inoculum density) in SH liquid medium augmented with various IBA concentrations. The growth of adventitious roots was determined based on biomass yield and relative growth rate (RGR) after 8 weeks of cultivation. Experimental results showed a significant (p ≤ 0.05) effect of IBA concentration on multiplication of adventitious roots in liquid medium. A significantly high root biomass yield (123.39 ± 7.11 g/L FW) and relative growth rate (RGR) (1.95 ± 0.03) was recorded in SH liquid medium at 4.92 µM IBA concentration after 8 weeks of cultivation (Table 2). In addition, the growth index (11.34 ± 0.71) (GI) was also found highest in same medium (Fig. 2a). However, the IBA concertation beyond 4.92 µM was not able to support further growth of adventitious roots. Furthermore, these results suggest that the multiplication of adventitious root in submerged condition was obtained in just half IBA concentration to that of induction medium (9.48 µM).
Table 2
Effect of different concentrations of IBA on biomass production in SH liquid medium after 8 weeks of incubation.
IBA (µM)
|
Fresh weight (g/L)
|
Dry weight (g/L)
|
Relative growth rate
|
Control
|
21.03 ± 1.18a
|
2.56 ± 0.26a
|
1.19 ± 0.02a
|
0.49
|
61.96 ± 4.84b
|
3.74 ± 0.26b
|
1.64 ± 0.04b
|
2.46
|
84.44 ± 5.79c
|
5.49 ± 0.31c
|
1.78 ± 0.03c
|
4.92
|
123.39 ± 7.11e
|
7.44 ± 0.44d
|
1.95 ± 0.03d
|
9.84
|
107.47 ± 7.29d
|
6.90 ± 0.43d
|
1.88 ± 0.04d
|
19.69
|
67.89 ± 4.79b
|
4.99 ± 0.32c
|
1.69 ± 0.03b
|
Values within the column followed by different letters significantly different at p ≤ 0.05 level as determined using Duncan’s multiple range test. A value represents mean ± standard error. |
Contrary to present results, Kannan et al. (2020) observed higher adventitious roots induction potential (i.e. 134.33 root number per explant) from leaf explants of Morinda coreia on ½ strength MS media augmented with 1.0 mg/L IBA, whereas, maximum proliferation efficiency of induced roots (i.e. 1.568 g/L FW, 5.082 growth ratio and 0.163 g/L DW) were attained on liquid MS medium enriched with same IBA concentration. Similarly, Wu et al. (2006) also reported induction of Echinacea angustifoli adventitious roots from root explants on half strength liquid MS media supplemented with IBA (1.0 mg/L), however, growth of induced root was maximum (11.8 g/L FW and 6.0 growth rate) in same strength liquid media fortified with 2.0 mg/L IBA. They also observed negative impact on root biomass growth beyond the 2.0 mg/L IBA. From the above results it can be deduced that 4.92 µM IBA in SH media is optimum for maximum adventitious root biomass accumulation under in vitro condition.
Effect of medium strength on multiplication of adventitious roots in submerged cultivation
In general, medium type and their elemental composition found to influence growth as well as overall productivity of in vitro plant cultures. Different strength of liquid SH medium (1/4X, 1/2X, 3/4X & 1X) supplemented with best responsive IBA concentration (i.e. 4.92 µM) were investigated to maximize the yield of root biomass. A significantly (p ≤ 0.05) high roots biomass (126.40 ± 23.90 g/L) and relative growth rate (1.86 ± 0.09) was recorded in half strength (1/2X) SH medium (Table 3) as compared to other medium strength tested in the study. GI was also highest in half strength (1/2X) SH medium (Fig. 2b). SH medium having 1/4x concentration exhibited slowest growth as evident from the root biomass yield. From the above results, it can be deduced that optimal concentration of macro and micronutrients in media seems to be the determining factor for in vitro induced adventitious root growth.
Table 3
Differential response of adventitious root multiplication on varying strength of SH liquid medium supplemented with IBA (4.92 µM) after 8 weeks of incubation.
Strength of SH media
|
Fresh weight (g/L)
|
Dry weight (gm/L)
|
Relative growth rate
|
1/4
|
97.87 ± 16.12ab
|
7.74 ± 1.14ab
|
1.77 ± 0.08ab
|
1/2
|
126.40 ± 23.90c
|
9.76 ± 1.64b
|
1.86 ± 0.09b
|
3/4
|
63.20 ± 13.11a
|
5.17 ± 1.00a
|
1.57 ± 0.08a
|
1/0
|
106.40 ± 15.04ab
|
9.71 ± 1.58b
|
1.83 ± 0.07b
|
Values within the column followed by different letters significantly different at p ≤ 0.05 level as determined using Duncan’s multiple range test. A value represents mean ± standard error. |
Corroborating to our results, adventitious root induced from Echinacea angustifolia showed maximum biomass yield in half strength MS medium supplemented with IBA (2.0 mg/L) as compared to 0.25, 0.75, 1.00, 1.50 and 2.00 strength (Wu et al. 2006). Similarly, half strength of MS medium fortified with IBA (3.0 mg/L) also found optimum for multiplication of adventitious roots induced from rhizome of P. hexandrum (Rajesh et al. 2014). Whereas, 1/4x strength of MS nutrients in combination with IBA (1.0 mg/L) resulted in maximum biomass accumulation in Periploca sepium adventitious roots (Zhang et al. 2011). It is well known that strength of the medium plays crucial role in growth and development of plant cells/tissues during cultivation under in vitro condition. The low salt concentration in the medium also increase the availability of nutrient ions (George et al. 2008).
Influence of various concentrations of sucrose on multiplication of adventitious roots in submerged cultivation
In plant cell and tissue cultures, sucrose is the principal carbohydrate used as energy source for growth and development. It is catabolized into glucose and fructose and it’s absorption rate varies with type of cultures (George et al. 2008). In the present work, diverse sucrose (1 to 5 % w/v) concentration in optimized media i.e. ½ strength SH media having 4.92 µM IBA were tried in submerged cultivation to improve further V. jatamansi adventitious roots multiplication. Among different concentrations of sucrose tested, a significantly high (p ≤ 0.05) root biomass (144.09 ± 11.36 g/L FW) and relative growth rate (2.01 ± 0.04) was obtained in medium augmented with 2% (w/v) sucrose after cultivation of 8 weeks (Table 4). Also, same media showed highest growth index (13.41 ± 1.14) as compared to other concentration (Fig. 2c). However, the root biomass yield on dry weight basis depicted dissimilar pattern with subsequent increase in sucrose concentration (4–5%). It may be due to formation of more extracellular polysaccharides with respect to the higher concentration of sucrose in the medium (Saiman et al. 2012).
Table 4
Effect of different concentrations of sucrose in ½ strength SH liquid medium supplemented with IBA (4.92 µM) on biomass production after 8 weeks of incubation.
Sucrose (%)
|
Fresh weight (gm/L)
|
Dry weight (gm/L)
|
Relative growth rate
|
1.0
|
100.30 ± 9.81b
|
6.63 ± 0.69a
|
1.84 ± 0.05b
|
2.0
|
144.09 ± 11.36c
|
8.91 ± 0.69b
|
2.01 ± 0.04d
|
3.0
|
136.81 ± 14.46c
|
9.59 ± 0.68b
|
1.98 ± 0.05bc
|
4.0
|
100.57 ± 8.65b
|
12.31 ± 0.56c
|
1.85 ± 0.04b
|
5.0
|
58.47 ± 9.69a
|
8.28 ± 0.88ab
|
1.58 ± 0.06a
|
Values within the column followed by different letters significantly different at p ≤ 0.05 level as determined using Duncan’s multiple range test. A value represents mean ± standard error. |
Similar to the present observations, Murthy and Praveen (2013) reported maximum accumulation of W. somnifera adventitious root biomass (113.58 g/L FW and 8.70 growth ratio) on half strength liquid MS media having 2% sucrose after four weeks of culture period. They also observed negative effect of increased sucrose concentration in media (3–8%) on accumulation of adventitious root biomass. In another study, 2% sucrose (w/v) concentration in MS medium was found optimum for highest yield of Gynura procumbens adventitious root biomass i.e. 13.8 ± 1.60 g FW per flask after four weeks of cultivation. Further, they also observed decrease in root biomass with further increase in sucrose concentration (7–10% w/v) in the media (Saiman et al. 2012).
It may be inferred from the results that 2 % sucrose in the ½ strength SH medium is optimum for the growth of V. Jatamansi adventitious root cultures under in vitro condition.
Determination of valerenic acid and its derivatives using Ultra Performance Liquid Chromatography analysis
Valerenic acid (VA) and its derivatives acetoxyvalerenic acid (AVA) and hydroxyvalerenic (HVA) are main active components of the herb V. jatamansi. In present study, the parent plant material (rhizome and leaves) and in vitro induced adventitious root samples were analysed for the identification and quantification of VA, AVA and HVA acid using UPLC-PDA. The yield of total valerenic acid derivatives (1525.14 ± 68.85 µg/g DW) was significantly (p ≤ 0.05) high in adventitious roots in comparison to parent plant parts i.e. rhizome (624.78 ± 13.67 µg/g DW) and leaves (200.17 ± 4.27 µg/g DW). However, valerenic acid (506.27 ± 10.34 µg/g DW) in rhizome was found higher as compared to leaves of the parent plant (79.23 ± 4.56 µg/g DW) as well as in vitro induced adventitious roots (70.66 ± 0.36 µg/g DW). Whereas, AVA content was significantly (p ≤ 0.05) high in adventitious root samples (534.91 ± 39.57 µg/g DW) than parent plant parts i.e. rhizome (118.51 ± 4.16 µg/g DW) and leaves (120.94 ± 7.48 µg/g DW). Similarly, Tousi et al. (2010) recorded higher fraction of VA (0.38 %), AVA (0.55%) and HVA (0.44%) in Valeriana officinalis adventitious roots induced from petiole explant on MS medium supplemented with IAA.
Interestingly, in vitro adventitious roots showed presence of HVA at significantly high amount (919.57 ± 28.85 µg/g DW), which otherwise not quantifiable in leaves as well as rhizome parts of the parent plants (Table 5 and Fig. 3). It is also pertinent to mention here that the comparative evaluation performed was between two-month (eight weeks) old in vitro adventitious roots versus rhizomes and leaves of approximately 2-year old plants grown under conventional agricultural cultivation condition. Thus, results of present study clearly suggesting the potential of V. jatamansi adventitious roots as a good alternative source of valerenic acid and its derivatives. In addition, in vitro induced adventitious roots could be a novel source of hydroxyvalerenic acid that was unquantifiable in parent plant parts.
Table 5
Quantitative analysis of valerenic acid, acetoxy valerenic acid and hydroxyl valerenic acid from plant part and adventitious root samples of V. jatamansi using Ultra Performance Liquid Chromatography (UPLC-PDA) system.
Sample
|
Metabolite content (µg/gm DW)
|
VA
|
AVA
|
HVA
|
Total valerenic acid derivatives yield
|
Rhizome
|
506.27 ± 10.34b
|
118.51 ± 4.16a
|
0.00 ± 0.00*a
|
624.78 ± 13.67b
|
Leaves
|
79.23 ± 4.56a
|
120.94 ± 7.48a
|
0.00 ± 0.00*a
|
200.17 ± 4.27a
|
Adventitious roots
|
70.66 ± 0.36a
|
534.91 ± 39.57b
|
919.57 ± 28.85b
|
1525.14 ± 55.20c
|
*NQ: Not quantifiable. Values within the column followed by different letters significantly different at p ≤ 0.05 level as determined using Duncan’s multiple range test. A value represents mean ± standard error. |
VA: Valerenic acid; AVA: Acetoxyvalerenic acid; AVA: Hydroxyvalerenic acid. |
Earlier, Singh et al. (2006) quantified valerenic acid in rhizome of V. officinalis and V. jatamansi plants by High performance thin layer chromatography (HPTLC). They found relatively higher valerenic acid content (0.42%) in V. officinalis than V. jatamansi (0.12% valerenic acid). Similarly, Srivastava et al. (2010) reported presence of valerenic acid (0.11%) in V. jatamansi rhizomes using HPTLC methods. Furthermore, Batra et al. (2016)found relatively low valerenic acid content (0.001%) in rhizomes of V. jatamansi. There is very limited information available on tissue culture raised V. jatamansi plants or tissues.
Recently, Partap et al. (2020) studied the effect of methyl jasmonate and yeast extract on accumulation of valerenic acid derivative from leaves and roots of nursery-grown, aeroponic and pot cultivated V. jatamansi plants. They reported maximum amount of VA (4.19 mg/g) in roots of plants grown under pot cultivation followed by nursery grown (3.18 mg/g) and aeroponic cultivation (1.78 mg/g). AVA content was found higher (2.38 mg/g) in roots grown under aeroponic condition.
Determination of essential oil, patchouli alcohol and other derivatives using Gas Chromatographic – Mass Spectroscopic (GC – MS) analysis
Valeriana jatamansi plant parts (rhizome & leaves) and adventitious root (1000g each, FW) were used for the extraction of essential oil using hydro-distillation method in a Clevenger apparatus. The plant rhizome yielded relatively higher essential oil content (0.4% v/w) as compared to in vitro induced adventitious root (0.059% v/w). However, trace amount of essential oil was obtained from the leaves of parent plants.
Earlier, Singh et al. (2013) reported a yield of 0.1–0.5% essential oil from two-year-old V. jatamansi plants fresh rhizomes cultivated in western Himalayas. However, comparatively high essential oil content (0.60–1.66%) was reported in thirteen V. jatamansi chemotypes collected from various agro-climatic zones of Himachal Pradesh, India (Singh et al. 2013, 2015). Previously, Sati et al. (2005) reported extraction of essential oil (0.03%) through steam distillation method from Valeriana wallichii leaves collected from Western Himalaya. Also, Coassini and Moneghini (1989) reported 0.1–0.45% essential oil yield from fresh leaves collected from three populations of Valeriana officinalis.
In present study, adventitious root induced from leaves yielded 0.059% v/w essential oil, which is reasonably low as compared to rhizomes harvested from field grown parent plants. However, it is worth mentioning that in vitro cultivation period of adventitious root is significantly low (2 months) as compared to field conditions (2 years).
Furthermore, the essential oil extracted from different samples was analysed by GC-MS to characterize its individual constituents. In this regard, a total of thirty-one phytochemical constituents were characterized and identified (Table 6, Fig. 4). These constituents represent 98.15%, 79.11% and 96.56% of essential oil obtained from rhizome, leaves and adventitious root samples, respectively. Overall, the GC-MS analysis exhibited presence of nine common constituents i.e. patchouli alcohol, α – patchoulene, β – patchoulene, seychellene, cis-β- guaiene, trans-β – guaiene, α – selinene, kessane and veridiflorol in essential oil extracted from parent plant parts (rhizome and leaves) and adventitious root samples. However, the percentagewise contribution (Fig. 5) of individual constituent in essential oil was patchouli alcohol > seychellene > trans-β – guaiene > veridiflorol > α- patchoulene of rhizome, patchouli alcohol > n - valeric acid > α-trans-bergamotene > seychellene > veridiflorol of leaves and patchouli alcohol > trans-β – guaiene > seychellene > α - gurjunene > α- guaiene of in vitro induced adventitious roots, respectively.
Table 6
Extraction and analysis of essential oil, patchouli alcohol and other derivatives using Gas Chromatographic – Mass Spectroscopic (GC – MS) system.
S.No.
|
Phytochemical constituents
|
Retention time (RT)
|
Retention index (RI)
|
Plant sample
|
Adventitious roots
|
Rhizome
|
Leaves
|
1.
|
n - Valeric acid
|
5.771
|
953
|
-
|
15.78
|
-
|
2.
|
Hexenyl isovalerate
|
15.197
|
1236
|
-
|
1.79
|
-
|
3.
|
Hexyl Isovalerate
|
15.425
|
1242
|
-
|
0.93
|
-
|
4.
|
β - Patchoulene
|
20.539
|
1386
|
1.72
|
0.98
|
0.42
|
5.
|
β - Elemene
|
20.68
|
1390
|
0.76
|
-
|
2.12
|
6.
|
Pentanoic acid
|
20.778
|
1393
|
-
|
1.06
|
-
|
7.
|
α-trans-Bergamotene
|
22.124
|
1434
|
-
|
8.31
|
4.08
|
8.
|
α - Guaiene
|
22.229
|
1438
|
2.49
|
-
|
6.19
|
9.
|
Aromadendrene
|
22.492
|
1446
|
1.89
|
1.02
|
-
|
10.
|
Seychellene
|
22.708
|
1452
|
6.34
|
4.43
|
9.52
|
11.
|
α - Gurjunene
|
22.751
|
1454
|
-
|
-
|
6.31
|
12.
|
α - Humulene
|
22.914
|
1458
|
1.19
|
-
|
3.63
|
13.
|
α - Patchoulene
|
23.116
|
1464
|
2.54
|
1.19
|
3.98
|
14.
|
Curcumene
|
23.671
|
1481
|
-
|
2.59
|
-
|
15.
|
α - Selinene
|
22.242
|
1497
|
0.86
|
-
|
-
|
16.
|
Caryophyllene
|
23.983
|
1490
|
-
|
-
|
0.41
|
17.
|
α - Bisabolene
|
-
|
1493
|
-
|
-
|
0.2
|
18.
|
Chamigrene
|
24.204
|
1496
|
-
|
-
|
2.82
|
19.
|
cis-β- Guaiene
|
24.291
|
1499
|
1.41
|
1.96
|
2.47
|
20.
|
trans-β - Guaiene
|
24.554
|
1507
|
4.42
|
0.82
|
17.82
|
21.
|
α - Selinene
|
24.975
|
1521
|
1.56
|
0.83
|
2.5
|
22.
|
Kessane
|
25.288
|
1532
|
1.32
|
1.01
|
0.85
|
23.
|
Epiglobulol
|
26.587
|
1573
|
2.42
|
-
|
5.26
|
24.
|
Isopatchoulane
|
27.104
|
1589
|
1.67
|
-
|
-
|
25.
|
β - Gurjunene
|
27.328
|
1596
|
-
|
1.9
|
1.3
|
26.
|
Veridiflorolol
|
27.359
|
1603
|
0.67
|
-
|
-
|
27.
|
Geranyl isovalerate
|
27.494
|
1601
|
-
|
2.93
|
-
|
28.
|
Longipinanol
|
27.686
|
1608
|
-
|
1.34
|
-
|
29.
|
Humulene epoxide II
|
27.815
|
1612
|
1.17
|
-
|
-
|
30.
|
Veridiflorol
|
29.196
|
1659
|
2.96
|
3.81
|
2.68
|
31.
|
Patchouli alcohol
|
29.671
|
1675
|
62.76
|
26.43
|
24.00
|
32.
|
Essential oil content (FW)
|
-
|
-
|
0.400 %
|
*TA
|
0.059 %
|
*TA: Trace amount. |
In accordance with present investigation, patchouli alcohol (48.47–65.04%) was reported to be the major constituent of essential oil extracted from rhizomes of V. jatamansi chemotypes obtained from Western Himalayas (Singh et al. 2013). Recently, a comprehensive study was performed to assess the effect of phenological stages and altitude on various constituents of essential oil extracted from V. jatamansi rhizomes cultivated in North-West Himalayas (Jugran et al. 2020). A quite significant variation in various constituents was reported with respect to phenological stage as well as altitude, however, patchouli alcohol (36.52–52.68%) was found to be the major component followed by β-patchoulene (1.14–2.70%), α-guaiene (0.28–5.08%), δ – guaiene (0.86–9.49%) and seychellene (1.06–4.56%) including various other minor molecules.
Contrary to present results, Das et al. (2011) reported maaliol (26.1%) as a major component followed by patchouli alcohol (9.3%) in fresh rhizomes of V. jatamansi collected from North-East Himalayas. This variation in the constituents of V. jatamansi essential oil can be attributed to the age of plants, time of harvest and geographical differences. However, it is quite clear from the results of present investigation as well as above discussion that patchouli alcohol is one of the major volatile component of V. jatamansi essential oil.
Determination of phenolic acids derivatives using Ultra Performance Liquid Chromatography analysis
Phenolic acids derivatives i.e. gallic acid, p-coumaric acid, rutin, ferulic acid, cinnamic acid and kaempferol were determined from plant rhizome, leaves and adventitious root using Ultra Performance Liquid Chromatography (UPLC-PDA) system. In vitro raised adventitious roots reported significantly higher phenolic acid derivatives (451.58 µg/g) as rhizome (187.79 µg/g) and leaves (263.68 µg/g) parts of the plant. In particular, adventitious root also showed significantly higher content of gallic acid (34.24 ± 1.20 µg/g), p-coumaric acid (162.46 ± 8.68 µg/g), rutin (217.86 ± 0.32 µg/g), ferulic acid (6.65 ± 0.12 µg/g) and kaempferol (22.82 ± 8.36 µg/g) as compared to parent plant part rhizome gallic acid (33.14 ± 2.90 µg/g), rutin (138.32 ± 1.91µg/g) and ferulic acid (5.39 ± 0.11 µg/g), whereas plant leaves showed gallic acid (33.41 ± 4.04 µg/g), p-coumaric acid (161.90 ± 8.09 µg/g), rutin (49.78 ± 0.13 µg/g), ferulic acid (0.72 ± 0.12 µg/g) and kaempferol (17.87 ± 1.85 µg/g). it was also observed that rhizome part does not showed detectable amount of p-coumaric acid and kaempferol, whereas leaves does not showed presence of cinnamic acid (Table 7; Fig. 6).
Table 7
Determination of phenolic acids derivatives and DPPH activity in plant parts and adventitious roots.
Plant sample
|
Phenolic acid derivatives (µg/g)
|
DPPH activity (%)
|
Gallic Acid
|
p-coumaric acid
|
Rutin
|
Ferulic acid
|
Cinnamic acid
|
Kaempferol
|
Total Phenolic acid derivatives
|
Rhizome
|
33.14 ± 2.90a
|
0.00 ± 0.00a
|
138.32 ± 1.91b
|
5.39 ± 0.11b
|
10.95 ± 4.16c
|
0.00 ± 0.00a
|
187.79 ± 1.81a
|
85.78 ± 1.62b
|
Leaves
|
33.41 ± 4.04b
|
161.90 ± 8.09b
|
49.78 ± 0.13a
|
0.72 ± 0.12a
|
0.00 ± 0.00a
|
17.87 ± 1.85b
|
263.68 ± 0.28b
|
61.98 ± 0.97a
|
Adventitious roots
|
34.24 ± 1.20c
|
162.46 ± 8.68c
|
217.86 ± 0.32c
|
6.65 ± 0.12c
|
7.82 ± 5.77b
|
22.82 ± 8.36c
|
451.85 ± 0.85c
|
87.09 ± 0.63b
|
Values within the column followed by different letters significantly different at p ≤ 0.05 level as determined using Duncan’s multiple range test. A value represents mean ± standard error. |
In addition, rhizome part recorded higher amount of cinnamic acid (10.95 ± 4.16 µg/g) and compared to adventitious root (7.82 ± 5.77 µg/g) (Table 7). Theses bioactive compounds have antioxidant, antimaicrobial, anticancer, anticholesterolemic acid and adventitious root culture could be alternate route for their biosysnthesis and production. In earlier studies, Bhatt et al. (2012) determined phenolic compounds i.e. gallic acid, hydroxybenzoic acid, caffeic acid, catechin, chlorogenic acid p-coumaric acid in different parts of planted and wild cultivated V. jatamansi plants. They reported significantly higher content of caffeic acid (158.56 mg/100 g) and hydroxybenzoic acid (390.58 mg/100 g DW) in planted condition, whereas, wild cultivated V. jatamansi plants recorded higher content of gallic acid (8.70 mg/100 g), coumeric acid (2.89 mg/100 g), chlorogenic acid (5.52 mg/100 g) and catechin (229.59 mg/100 g). In addition, aerial portion of wild cultivated V. jatamansi plants revealled higher content of gallic acid, p-coumaric acid, chlorogenic acid and catechins. However, caffeic acid and hydroxybenzoic acid content was found higher in aerial portion of planted V. jatamansi plants. In another study, Jugran et al. (2020) observed significant variations in different phenolic i.e. gallic acid, p-coumaric acid, chlorogenic acid, catechin, caffeic acid and hydroxy-benzoic acid in pre-flowering, flowering and post-flowering stages of V. jatamansi plant population wrt their occurance in high, intermediate and low altitude. Gallic acid content was found to be highest (9.39 mg/100 g DW) in pre - flowering stage of plant population of low altitude region. p-Coumaric acid content was maximum (24.34 mg/100 g DW) at high altitude during pre-flowering stage of the plant. In case of chlorogenic acid, post-flowering stage showed highest (7.45 mg/100 g DW) content at high altitude. Catechin content was maximum (6.03 mg/100 g DW) in post-flowering stage of plant population at high altitude conditions. Caffeic acid and hydroxy-benzoic acid was both detected highest (2.61 & 6.24 mg/100 g DW) in pre-flowering stage of plant population collected from intermediate altitudinal region. Presence of above bioactives compound in adventitious root culture showed an alternate source for their production on sustainable basis for medicinal uses.
Analysis of DPPH (2, 2-diphenyl-1-picrylhydrazyl) activity
Comparative analysis of antioxidant activity using DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay was done using methanolic extracts of rhizome, leaves and adventitious roots samples. The data revealed that adventitious root sample (87.09 %) showed higher free radical scavenging activity as compared to rhizome (85.78 %) and leaves (61.98 %) samples (Table 7). However, leaf induced adventitious root shows higher DPPH activity with leaf samples of parent plant. DPPH generally used to evaluate the free radical-scavenging activity of natural antioxidants from the plant and this free radical activity trigger production of marker compounds and act as major factor in biological damages during different stages of culture (Boo et al. 2018). However, DPPH activity in the plant also reflect the stress levels confronted by the tissues. Adventitious root of H. perforatum showed a steady increase in the free radical scavenging activity (Cui et al. 2011) and are specific to plants. The results of present investigation are in agreement with report of Boo et al. (2018), where a proportional increase (10 mg mL-1) in concentration of free radical scavenging activity was found higher than 80% in adventitious root culture of Platycodon grandiflorum.
Effect of culture stages on production of valerenic acid derivatives
In general, in vitro plant cell and tissue culture shows variability in growth as well as metabolite production at various culture stages. Therefore, production of valerenic acid derivatives at different culture stages i.e. a) induced from leaf explants on semi - solid SH media + 9.84 µM IBA (P0), b) multiplied on semi - solid SH media + 4.92 µM IBA (P1) and c) submerged cultivation in liquid SH media + 4.92 µM IBA (P2) of V. jatamansi adventitious root development was also studied in present investigation. The results showed presence of VA, AVA and HVA in all the stages of adventitious root formation and multiplication (Fig. 7). Total valerenic acid derivatives yield were significantly (p ≤ 0.05) increased (302.28–1625.98 µg/g DW) from culture stage 1 to stage 3. In case of individual metabolite, there was significant (p ≤ 0.05) enhancement in AVA (92.51–620.72 µg/g DW) and HVA (140.42–933.95 µg/g DW) content with respect to different culture stages (Fig. 7). From the trend, it can be deduced that in culture stage 1 the priority of the tissue is specifically towards its growth and development, whereas during stage 2 and 3 the high metabolic yield indicates the shift or activation of secondary metabolism. Also, there might be a possibility that submerged cultivation create more stressing environment, thus enhancing biosynthesis of valerenic acid derivatives, especially in stage 3 cultures.
Similar information on secondary metabolite production in relation to in vitro plant tissue culture stages/passage is very limited. Hagimori et al. (1980) quantified digoxin content in passage one and second of callus induced from seedlings and leaf disk of Digitalis species inoculated on different media under light and dark condition. The induced callus showed diverse tendency of metabolic profile in first and second passage of culture. Callus induced from seedling showed high digoxin content in first passage. Whereas, in second passage digitoxin content was not detected. However, leaf disc induced callus exhibited similar metabolite content in first passage as in inoculum. In contrast, callus proliferated during second passage revealed varying digoxin content in different Digitalis species. In another study on effect of passage on in vitro shoot multiplication of Cassia angustifolia, Siddique et al. (2015) studied the impact of TDZ on shoot cultures up to 5th passages of sub-culture in MS medium. They observed continuous increase in shoot number (12.0 ± 0.9) and length (6.9 ± 0.29 cm) up to fifth passages on MS medium fortified with 5.0 µM TDZ. In the current research work, the aim was to determined metabolite content in adventitious roots of V. jatamansi at different passage times.
Similarly, Garica-Mateos et al. (2005) reported significant accumulation of alkaloids i.e. alpha (43.26–45.58%) and beta (49.75–52.44%) erythroidines up to fifth sub-culture, after that is decrease trend. The erysodin and erysovin, content was only quantified in the seventh sub-culture of cotyledons induced callus culture. In addition, Coppede et al. (2014) quantified higher amount of Quinonemethide triterpenoid (QMTs) i.e. maytenin (1,147.90 µg/g) and 22b-hydroxymaytenin (1,032.89 µg/g) in leaf induced callus of Maytenus ilicifolia after twelve days of inoculation. However, during subsequent subculturing from sixteen to forty-eight days, it showed decreases pattern of QMTs.
Also, Le et al. (2019) reported a consistent increase in ginsenosides i.e., protopanaxadiol and protopanaxatriol during long (twenty-year old) and short-term (one-year old) cultivation of Panax ginseng adventitious roots. The accumulation of secondary metabolites under in vitro condition significantly affected by numbers of factors i.e. explants, selection of genotypes, friability, somaclonal variations, repetitive subculturing for a longer time, morphological variations, DNA methylation and genetic stability (Bourgaud et al. 2001; Fu et al. 2012; Coppede et al. 2014). The above facts clearly stated the significant role of culture stages on accumulation of metabolite content under in vitro condition.
Overall process of induction, multiplication and submerged cultivation
The complete process of V. jatamansi adventitious root culture cultivation can be divided into three stages; namely, adventitious root induction, multiplication and submerged cultivation for scale up production (Fig. 8). The detailed bioprocess can be summarized under following heads:
Adventitious root induction: In this stage, adventitious roots were induced from leaf explants on optimized SH media fortified with 9.84 µM IBA within eight days of inoculation under aseptic condition.
Multiplication of adventitious roots: After four weeks of induction, induced roots were further amplified on semi-solid ½ strength SH media having 4.92 µM IBA and 2.0 % sucrose.
Submerged cultivation of adventitious roots: Considering the development of alternative route for production of valerenic acid derivatives, large-scale multiplication of adventitious roots was done through submerged cultivation in optimized ½ strength liquid SH media enriched with 4.92 µM IBA and 2.0 % sucrose for two months (eight weeks) under in vitro condition.
Considering the developed
in vitro protocol, the complete process took two months (eight weeks) after induction of adventitious roots from leaf explants
of
V. jatamansi. Whereas, the generation of commercially valued rhizomes or root biomass through conventional means generally takes 2 years after transplanting in field condition (Singh et al. 2010). In addition, the conventional cultivation gets jeopardised by slow germination, poor viability and long dormancy of seeds as well as limited planting material through vegetative propagation (Rana et al. 2004). These issues not only affecting the availability of quality raw material to herbal industries on sustainable basis, but also affecting the plant population in its natural habitat. The feasibility of submerged cultivation bioprocess as compared to conventional cultivation can be assessed on the basis that
in vitro adventitious roots can yield over two times higher essential oil (Table 8).
Table 8
Comparative analysis of essential oil and total valerenic acid derivative yield in conventional and submerged cultivation.
Type of cultivation
|
Cycle
(Month)
|
Essential oil yield (%)
|
Total valerenic acid derivative (µg/g)
|
Yield/two year
|
Essential oil (%)
|
Total valerenic acid derivative (µg/g)
|
Conventional*
|
24
|
0.500
|
624.78
|
0.500
|
624.78**
|
In vitro adventitious root (submerged cultivation)
|
02
|
0.059
|
1525.14
(one cycle)
|
0.708***
(6x2 year = 12 cycle)
|
18301.68***
(6x2 year = 12 cycle)
|
*Singh et al., 2010. |
** quantified during present investigation |
***estimated figures |