Journal of Medicinal Plants Research Vol. 5(23), pp. 5528-5533, 23 October, 2011 Available online at http://www.academicjournals.

org/JMPR ISSN 1996-08752011 Academic Journals

Full Length Research Paper

Growth, herb yield and phytochemical contents in a medicinal herb Andrographis paniculata under saline irrigation
I. Rajpar1*, Y. M. Khanif2, Zia-ul-hassan1, A. N. Shah1, M. Arshad3 and S. Galani4
Sindh Agriculture University, Tandojam, Pakistan. 2 Universiti Putra Malaysia, Malaysia. 3 Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, Karachi, Pakistan.
Accepted 22 August, 2011
1

Andrographis paniculata (King of bitters/Hempedu Bumi) is a traditional medicinal herb of family Acanthaceae. It is commonly used to cure a variety of ailments and diseases. This study was designed to evaluate the herb yield and phytochemical contents in two germplasm accessions of Malaysian A. paniculata, viz. 11261 and 11265, grown with different quality of irrigation water, that fresh (ECiw 0.12 dS m-1) and saline (ECiw 2.5 and 5.0 dS m-1). Just before blooming, the plants were harvested to record data on six basic traits, including plant height, length and width of main-stem leaves, length and number of -1 -1 + + primary branches plant and dry herbage yield (plant ). Concentrations of Na and K , coupled with + + K /Na ratio, were determined in the cell sap extracted from the top fully matured leaves. Phytochemicals, viz. Andrographolide (AG), Neoandrographolide (NAG) and 14-deoxy-11, 12didehydroandrographolide (DDAG), were determined in the dry matter of aerial parts using highperformance liquid chromatography (HPLC). No significant changes in plant growth and dry herbage yield (plant-1) were observed with 2.5 ECiw (dS m-1) water, compared to 5.0 ECiw water, where reductions in same indices were >13% and statistically significant. Adverse effects of water salinity on growth and dry herbage yield can be attributed with significantly higher concentration of Na+ and lower + + + concentration of K and lower K /Na ratio in the leaf sap. Phytochemical contents in dry matter were generally unaffected by water salinity. With the exception of Na+ concentration in leaf sap, response of both accessions to saline irrigation was similar. These results suggest that both accessions exhibited good potential to withstand to salty water environment and produce considerably high amount of phytochemicals under both normal as well as saline irrigation conditions. Key words: Andrographis paniculata, medicinal plants, phytochemicals, salinity. INTRODUCTION Most of the agriculturally productive area falls in arid and semi-arid regions of the world, where irrigation water is imperative for successful crop production (Ibrahim et al., 2006). Unfortunately, there is a severe shortage of good quality water in arid lands (Kataria, 2008) and to augment inadequate water supply, poor quality water (Zahid et al., 2003) within certain limits and with intelligent management is being exploited and utilized (Latif and Beg, 2004) in crop production. Currently, cultivation of plants with saline water having physiology for moderating the ill effect of salts seems to be an effective and intelligent management practice in saline irrigated agriculture (Ibrahim et al., 2006). Researchers have identified more than 1500 salttolerant plant species in the world (Qureshi and BarretLennard, 1998). Since medicinal and aromatic plants are gaining significant importance in the world (Hussain et al., 2003), there is a lack of systematic information on physiology of medicinal and aromatic plants under saline irrigation conditions. Although, there are several aromatic and medicinal valued plant species with tremendous potential to tolerate salts stress (Hussain et al., 2003).

*Corresponding author: inayatullahrajpar@gmail.com.

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Some traditional medicinal herbs still need to be evaluated for salt-tolerance. Among traditional medicinal species, Andrographis paniculata (King of bitters /Hempedu Bumi) is an herb of Acanthaceae family commonly used to cure many ailments and diseases (Kumar et al., 2004). Every part of the plant has its therapeutic value. Especially leaves (fresh and dry) of A. paniculata are extensively used in Asian traditional medicines and in various herbal combinations (Srivastava et al., 2004). The plant extracts exhibits antityphoid, antihepatotoxic, antimalarial, antibiotic, antihepatitic and antiinflamatory effects. The therapeutic activities in this plant are due to the Andrographolide (AG) and its related diterpene lactones such as Neoandrographolide (NAG) and 14-deoxy-11,12-didehydroandrographolide (DDAG) (Srivastava et al., 2004) and DDAG. In recent studies, AG has shown activity against HIV virus in vitro (Calabrese et al., 2000). Neoandrographolide has also shown activity against malaria (Misra et al., 1992) and various liver disorders (Kapil et al., 2001). Although, this plant grows on a wide variety of habitats such as mountain slopes, rangelands, wetlands, seashores and even on roadside, etc. Its physiology under saline conditions needs to be evaluated. Hence in this study, we compared growth, dry herbage yield and phytochemical contents of two A. paniculata germplasm accessions under saline irrigation conditions.

Quik Chem, FIA+ 8000 Series). Fertilizer management To ensure adequate nutrient supply, 40 kg of each N, P2O5 and K2O ha-1 was applied as a basal dose to each pot using NPK fertilizer containing 20% N, 20% P2O5 and 20% K2O. Preparation and application of saline water Immediately after transplantation, plants in pots were watered for six days with ordinary tap water. Salinization commenced 7 days after transplantation. There were three replicate pots of each of 3 saline water treatments per accession. Saline water treatments consisted of: SW 0 (continuous watering with fresh water of ECiw 0.12 dS m-1 and sodium adsorption ratio (SAR) 0.742), SW 1 (watering with saline water having 2.5 ECiw (dS m-1) and 25 SAR) and SW 2 (watering with saline water having ECiw 5.0 (dS m-1) and SAR 25). Saline water for each treatment was prepared by dissolving NaCl and CaCl2.2H2O in tap water at 20:1 ratio, following the method of Rowell (1994). Control plants in SW 0 treatment were maintained continuously by applying tap water, while in saline (SW 1 and SW 2) water treatments plants were irrigated with both saline and tap waters in cycle, that is, after every three saline water applications, plants in both saline water treatments were once irrigated with tap water. Electrical conductivity of water samples was determined using same digital meter as for soil. Sodium adsorption ratio was calculated from the concentrations of Na+, Ca2+ and Mg2+ (m molc l-1). Leaf sampling, extraction and chemical analysis of cell sap

MATERIALS AND METHODS Experimental conditions The experiment was conducted during November 2005 to February 2006 in a glasshouse equipped with natural ventilation, light and temperature at the Universiti Putra Malaysia.

For leaf sap extraction, two top fully matured leaves were separated from each plant just before blooming; samples were placed in micro-tubes and stored at -10C. Cell sap was extracted following the method of Gorham et al. (1997) and analyzed for K+ and Na+ concentrations using Auto-analyzer (Lachat Instruments, Quik Chem, FIA+ 8000 Series). Growth and yield variables

Raising of seedlings and transplantation Seedlings of both accessions were initially raised in the shed-house for two weeks by sowing water soaked (overnight) seed in plastic trays (36 x 26 cm surface and 10 cm deep) containing a mixture of soil and river sand (2:1). Later these seedlings were transferred into other trays of similar size filled with mixture (2:1:1) of soil, sand and peat (Pure peat, Greengress SDN. BHD. Malaysia). When the seedlings reached the required age (40 days after sowing), they were carefully removed from trays and transplanted into the soil filled earthen pots (22 x and 23 cm deep). Each pot contained two plants at a spacing of 8 cm. The soil placed in the pots was heavy textured clay soil of the Munchong (Tropeptic Haplorthox) series with 73% clay, 10.23% silt and 16.23% sand. The soil had 3.7% organic carbon (OC), 0.29% total nitrogen (N), 5.6 pH (after liming) and 0.8 dS m-1 electrical conductivity (ECe). The soil was limed with two tons of dolomite ha-1 a week before transplanting. Soil texture was determined by standard pipette method. Electrical conductivity and pH were determined in 1:1 soil water extract with electrodes using digital meter (Radiometer analytical, IONcheck65, Meterlab). Total carbon was determined by TOC-analyzer (Leco, Model CR-412). Total N was determined by Auto-analyzer (Lachat Instruments,

After leaf sampling, both plants from each pot were harvested by cutting at soil level. The data was recorded on plant height (cm), length and width (cm) of main-stem leaves and numbers and length (cm) of primary branches. Harvested plant samples were cut into small pieces and above ground dry herbage yield (plant-1) was determined by air-drying fresh plant tissues to constant weight using thin layer air-drying machine set at 50 with 1.5 ms-1 C velocity.

Phytochemical analysis Dried plant tissues were powdered in a stainless steel mill (IKA LABORTECHNIK, MF10 basic). Extract was obtained using analytical grade dichloromethane-methanol (1:1) solvents purchased from Fisher Scientific ((M) Sdn. Bhd.). The extract was filtered through Whatman No: 1 filter paper dried by rotary evaporator at 50 under reduced pressure and stored in a dry C state until analysis. Phytochemical determinations were performed using HPLC analysis. The HPLC system consisted of a Waters 2695 separation module, a mobile phase degasser, an auto sampler, a Waters 2487 UV detector and Empower Software (Waters, USA). The column used was a 150 x 4.6 mm, 3.5 M

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Table 1. Plant height, leaf width, leaf length, number of primary branches plant-1, and primary branch length of two Andrographis paniculata accessions (11261 and 11265) irrigated with fresh and saline water.

Saline water Plant height (cm) SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean Leaf width (cm) SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean Leaf length (cm) SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean

Accession 11261 57.83 56.50 50.33 54.88a 11265 57.50 55.66 51.16 54.77a

Mean

57.66a 56.08a 50.75b

3.500 3.150 2.666 3.105a

3.333 3.230 2.500 3.021a

3.416a 3.190a 2.583b

10.500 10.500 10.000 10.333a

10.500 10.333 8.833 9.833b

10.500a 10.417a 9.417b

Interaction effect was not significant. Means followed by a common letter in a column do not differ significantly at the 5% probability level by Tukey.

Agilent C18 column. The separation was carried out under isocratic elution with 2:1 (v/v) acetonitrile/phosphate buffer (5 mM sodium phosphate, adjusted to pH 3.2 with phosphoric acid) with 1% triethyamine. The flow rate was set at 0.8 ml/min, the wavelength was monitored at 225 nm, and the injection volume was set at 20 L. Statistical analysis All data were subjected to analysis of variance (ANOVA) using SAS statistical software version 8 (SAS Institute Inc., Cary, NC, USA) and means were compared using Tukeys honestly significant difference test (HSD) at alpha 0.05.

main-stem leaves, primary branches and dry herbage yield plant-1 (Tables 1 and 2) for both accessions examined. Plants irrigated with 5 ECiw water exhibited >13% reduction over control in most of the variables recorded. The reductions in the same variables at 2.5 ECiw level were < 7% and not significant (p > 0.05). The differences among accessions over all ECiw levels and salinity * accession interaction were not significant (p < 0.05). It appeared that the effect of water salinity was similar for both accessions in terms of width and length of leaves, length and number of primary branches and dry herbage yield plant-1. Effect of saline water on Na and K concentrations in leaf sap Results of Na and K analysis in leaf sap (Table 3) indicated that compared to control, plants irrigated with saline waters had higher concentration of Na+ and lower + concentration of K in their leaves, which resulted in lower + + K /Na ratio. As expected, higher concentration of Na+ in cell sap was the result of NaCl dissolved in irrigation water applied to plants. The lower concentration of K+ reflects the competitive inhibiting effect of Na+ for K+ under saline condition (Marschner, 1995; Ashraf and Orooj, 2006). Over all, water qualities significant (p < 0.05) differences were found among accessions for Na+ and K+ accumulation in leaf sap. The concentrations were considerably higher in accession 11265 than in 11261. It
+ + + +

RESULTS AND DISCUSSION Visual observations Typical visible salt injury symptoms resulting from Na+ toxicity like seedling mortality, lodging of the stems, variation in size, necrotic areas, and tip burn (USSL, 1954; Bains and Fireman, 1964) did not appear on both accessions across both water qualities during the study. This suggests that up to an ECiw 5 both accessions grew + well without showing any apparent evidence of Na and Cl toxicity or nutrient deficiency. Effect of saline water on growth and dry herbage yield Significant effect (p < 0.05) of water salinity was found on

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Table 2. Number of primary branches plant-1, primary branch length and dry herbage yield of two Andrographis paniculata accessions (11261 and 11265) irrigated with fresh (0.12 ECw) and saline (2.5 and 5 ECw) water.

Saline water Number of primary branches (plant ) SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean Primary branch length (cm) SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean Dry herbage yield (g plant-1) SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean
-1

Accession 11261 17.66 17.00 16.16 16.94a 11265 19.00 18.00 14.33 17.11a

Mean

18.33a 17.50a 15.25b

39.33 35.16 33.50 36.00a

38.83 37.00 31.83 35.88a

39.08a 36.08a 32.66b

10.236 9.382 6.447 8.687a

9.568 9.483 7.452 8.834a

9.880a 9.433a 6.960b

Interaction effect was not significant. Means followed by a common letter do not differ significantly at the 5% probability level by Tukey.

Table 3. Na+ and K+ concentrations (mol m-3) and K+/Na+ ratio in two Andrographis paniculata germplasm accessions (11261 and 11265) irrigated with fresh and saline water.

Saline water Na+ (molm -3) SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean K+ (molm -3) SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean K+/Na+ SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean

Accession 11261 50.286b 92.320b 188.676a 110.430b 11265 54.693c 137.536b 291.736a 161.320a

Mean

52.49c 114.93b 240.21a

101.193 95.73 82.33 93.09b

138.02 137.77 90.85 122.21a

119.60a 116.75a 86.60b

2.255 1.051 0.444 1.250a

2.171 1.001 0.313 1.162a

2.214a 1.025b 0.378c

Interaction effect was not significant. Means followed by a common letter do not differ significantly at the 5% probability level by Tukey.

should be noted that although the differences between accessions for growth and dry herbage yield per plant

were smaller and non-significant, adding NaCl to water showed marked difference among accessions in terms of

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Table 4. Aandrographolide, Neoandrographolide and 14-deoxy-11,12-didehydroandrographolide in two Andrographis paniculata accessions (11261 and 11265) irrigated with fresh and saline water.

Saline water Andrographolide (% DM) SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean Neoandrographolide (% DM) SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean 14-deoxy-11, 12-didehydrogegrapholide (% DM) SW0 (0.12 ECiw) SW1 (2.50 ECiw) SW2 (5.00 ECiw) Mean

Accession 11261 3.8600 3.9933 2.7333 3.5289a 11265 2.6933 3.3467 3.3667 3.1356a

Mean

3.2767a 3.6700a 3.0500a

2.0733 2.1067 1.7533 1.9778a

1.4967 1.4767 1.7967 1.5900a

1.7850a 1.7917a 1.7750a

0.2633 0.2533 0.1900 0.2355a

0.2066 0.2700 0.2166 0.2311a

0.2350a 0.2616a 0.2033a

Treatment, accession and interaction effects were not significant. Means followed by a common letter in a column do not differ significantly at the 5% probability level by Tukey.

Na+ and K+ accumulation. This might be due to the function of some genes involved in K+/Na+ discrimination under saline conditions (Gorham et al., 1997; Yamaguchi and Blumwald, 2005). Marked genotypic differences in accumulation of Na+ and depletion in K+ has long been demonstrated by numerous investigators (Amtman and Sanders, 1998; Rajpar and Wright, 2000). Lower (< 1) K+/Na+ ratio displayed by both accessions in 5 ECiw treatment indicates their similar response to salinity. Effect of saline water on phytochemicals (AG, NAG and DDAG) Saline water treatments had no significant (p > 0.05) effect on the contents of (Table 4) medicinally important phytochemicals (AG, NAG and DDAG). Though, the accession x salinity interaction was not significant in terms of phytochemical contents, compared to control, both accessions showed considerably higher values for all three phytochemicals at 2.5 ECiw level, whereas at ECiw 5 water salinity did not alter behavior of plants to produce phytochemicals. It is evident from this and other studies (USSL, 1954; Hamdy, 1996; Jatoi, 2005) that irrigation with saline water up to a certain degree is not necessarily adverse to some plants, rather higher yields may be obtained and sometimes even the quality of -1 produce can be better. No change at 80 mmol L NaCl -1 and sharp rise at 120 mmol L NaCl in proline concentration have recently been reported by Ashraf and

Orooj (2006) for a traditional medicinal plant named Ajwan (Trachyspermum ammi L.). Many plant species including traditional medicinal herbs (Zidan and Elewa, 1995; Bohnert and Jensen, 1996; Hajar et al., 1996; Chen and Murata, 2000; Chenusamy et al., 2005; Ashraf and Orooj, 2006; Kataria, 2008) accumulate organic (proline, betaine, polyoles, sugar alcohols and soluble sugars) osmolytes (osmoprotectants) to adjust osmotic stress. In the presented study, we found that at low water salinity, both accessions were able to produce consider amount of phytochemicals, whereas at maximum water salinity they maintained the concentrations of phytochemicals. Conclusions In conclusion, both the accessions of A. paniculata, viz. 11261 and 11265 exhibited good potential to withstand to salt water environment and produce considerably high amount of medicinally important phytochemicals, viz. Andrographolide (AG), Neoandrographolide (NAG) and 14-deoxy-11, 12-didehydroandrographolide (DDAG). Further validation of these results in field trials is warranted. ACKNOWLEDGEMENTS This study was supported by the Islamic Development Scholarship Program for the academic Year 1426/ 2005-

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2006 (FN:18/PAK-01/PD26). The phytochemical analysis was carried out in the Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, UPM with financial support from the Department of Land Managemnt, Faculty of Agriculture, Universiti Putra, Malaysia. We also thank Cancer Research and Drug Discovery (CRDD) group, Mr. Jamil and Ms. U.K. Asmaon for technical assistance.
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