Thai J. Pharm. Sci. 31 (2007) 91-99
91
Original article
HPLC determination of active diterpene lactones from Andrographis paniculata Nees planted in various seasons and regions in Thailand Chamnan Patarapanich1*, Suwanna Laungcholatan1, Nuntakan Mahaverawat2, Chaiyo Chaichantipayuth1 and Sunibhond Pummangura1 1Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2The Government Pharmaceutical Organization, Rama VI rd., Ratchatewi, Bangkok 10400. Thailand
*Corresponding author: E-mail address:
[email protected]
Abstract: A quantitative determination of four known diterpene lactone compounds : dehydroandroghapholide (C-2), andrographolide (C-3), neoandrographolide (C-4) and deoxyandrographolide-19-β-D glucoside (C-5) from the leaves of Andrographis paniculata, Nees. has been studied by using a reversed phase high performance liquid chromatographic technique. The system comprised with a µ-bondapak C18 column as stationary phase, a 42:58 mixture of methanol : water as mobile phase (flow rate 2 ml/min) and a UV-detector at wavelength of 255 and 220 nm. The relative retention time of each diterpene lactone were found to be 0.22 (C-3), 0.45 (C-5), 0.75 (C-2) and 1.00 (C-4). The linear standard curve of each compound was obtained in the following concentration range; C-2 (0.10-0.97 mg/ml, correlation coefficient (r) = 0.9990 and 0.9709), C-3 (0.10-0.42 mg/ml, r = 0.9998 and 0.9925), C-4 (0.10-0.30 mg/ml, r = 0.9998 and 0.9886) and C-5 (0.10-0.20 mg/ml, r = 0.9991 and 0.9917). The method was found to be highly accurate with low relative standard deviation as follows: 0.89% (C-2), 0.80% (C-3), 1.27% (C-4) and 1.67% (C-5). The contents of the four diterpene lactones in the sample of Andrographis paniculata collected from various regions in Thailand determined in terms of average percentage content from northern, southern, central & east and northeastern region, as follows: C-2 = 2.13 ± 0.96, 1.39 ± 0.32, 1.52 ± 1.00 and 1.81 ± 0.76, respectively; C-3 = 2.76 ± 0.82, 4.39 ± 1.02, 3.55 ± 1.69 and 3.24 ± 1.12, respectively; C-4 = 1.89 ± 0.44, 1.63 ± 0.37, 1.42 ± 0.59 and 1.57 ± 0.80, respectively; C-5 = 1.82 ± 1.07, 0.71 ± 0.54, 0.73 ± 0.4 and 1.07 ± 1.11, respectively. The contents of four diterpene lactones from the samples collected every month in one year-cycle were found in terms of the highest content and the lowest content as follows: C-2 = 7.30 ± 0.48% w/w (April), and 0.61 ± 0.58% w/w (December); C-3 = 6.02 ± 0.47% w/w (November) and 0.82 ± 0.86% w/w (February); C-4 = 2.02 ± 0.35% w/w (December) and 0.61 ± 0.58% w/w (April); C-5 = 3.81 ± 0.39% w/w (March) while C-5 content was too low to be detected (October and November). Moreover, the total diterpene lactone content in the leaves of Andrographis paniculata was found to be highest at 11.81% w/w in April and lowest at 7.68% w/w in October. Keywords: Andrographis paniculata Nees.; Andrographolide; Dehydroandrographolide; Deoxyandrographolide19-β-D-glucoside; Diterpenoid lactone; HPLC; Neoandrographolide; Quantitative determination; Region; Season; Thailand
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Introduction Andrographis paniculata Nees (AP), a well known small shrub belonging to the Acanthaceae family, is known in Thai name as Fah Talai Joan, Nam Lai Pangporn and Yaa Kanngu. This plant is popularly cultivated and is also easily propagated. All parts of the plant has bitter taste. The whole plant has been used for treatment of dysentery and fever in Mianmar and the People Replublic of China [1]. In Thailand, its leaves have been used for the treatment of tonsilitis [2], fever [3], dysentery and diarrhea [4], inflammatory injury [5], herpes simplex and herpes zoster infection [6]. In Southeast Asia, AP has been traditionally used as an antidote for snakes and insects poison and as an antimalarial drug [7]. Phytochemical studies have shown that the leaves of AP contain several diterpenoid lactones such as andrographolide [8], neoandrographolide, deoxyandrographolide- 19-β-D-glucoside, deoxyandrographolide [9] and dehydroandrographolide [10] together with other types of natural compounds. Numerous pharmacological activities of the AP extract have been reported including antimicrobial [11-13], anticoagulant [14], antifertility [15-16], anthelmintic [1718], hypotensive [19], antiinflammatory [20] and antispasmodic [21] activities. The biological activities of pure andrographolide have also been reported to be antispasmodic [22], antifertility [16] and anthelmintic [23] agents. For dehydroandrographolide has also been shown to possess the antispasmodic activity [24]. In term of toxicity, it has been reported that the aqueous extract of AP has an LD50 > 5000 mg/kg in rat [25], whereas the alcoholic extract has an LD50 > 1000 mg/kg in mice [12]. In addition, oral administration of andrographolide at 18 g/kg in rat appears no physical abnormality in the animal [25]. In Thailand the Office of Primary Health Care has promoted the use of AP for the treatment of common cold, sore throat and diarrhea. Meanwhile, the Ministry of Public Health has also promoted the plantation of this medicinal plants in each Community Hospital
C. Patarapanich et al. throughout the country for sufficient supply. However, concerns on variation in content and chemical profile caused by plantation conditions (geologic area, fertility of soils, climate, season, etc.), plant breeding and age at harvest have usually been raised. Therefore, in order to assure the effectiveness and the quality of the cultivated plants, it is necessary to monitor availability of the bioactive constituents both in terms of quantitative and qualitative aspects. This research work aimed to separate, identify, and quantitative determination of the bioactive diterpenoid lactones in the leaves of AP. These include : dehydroandroghapholide (C-2), andrographolide (C-3), neoandrographolide (C-4) and deoxyandrographolide19-β-D glucoside (C-5). The results obtained also determine from samples collected in various seasons and regions in Thailand. The data obtained would provide useful information for the cultivation of AP with respect to optimum time of harvest and area for plantation.
Materials and Methods Instruments Melting point apparatus (Electrothermal melting point apparatus), Infrared spectrophotometer (Shimadzu IR-400), Mass spectrophotometer (Jeol FÓ300 double focussing), HPLC, pump (Perkin Elmer series 410), integrator (Perkin Elmer series LCI-100), detector (Perkin Elmer series LC-235 Diode array). Reagents Solvents (ethanol, methanol, chloroform) and Silica gel 60 GF 254 TLC plates (20Ó20 cm and 20Ó5 cm), Silica gel G60 230-400 mesh, Silica gel G (0.040-0.063 mm) were perchased from Merck (Damstadt Germany), Reference Standards: dehydroandrographolide (C-2), andrographolide (C-3), neoandrographolide (C-4) and deoxyandrographolide-19-β-D-glucoside (C-5) were presented by Professor Lian Xiaotian of Institute of Materia Medica, Chinese Academic of Medical Sciences, Beijing, People Republic of China.
Thai J. Pharm. Sci. 31 (2007) 91-99 Plant materials The plant samples were randomly collected within the same period of time (first two weeks of October) from provinces in four different regions: northern region (1. Phitsanulok, 2. Lampang, 3. Nakhonsawan, 4. Phetchabun, 5. Chiangmai), central and eastern region (1. Nakhonnayok, 2. Suphanburi, 3. Phetchaburi, 4. Bangkok, 5. Chanthaburi, 6. Prachinburi), southern region (1. Prachuapkirikhan, 2. Suratthani, 3. Phuket, 4. Phangnga, 5. Ranong), northeastern region (1. Sakonnakhon, 2. Nakhonphanom, 3. Surin, 4. Khonkaen, 5. Kalasin). The collected samples were dried in a hot air oven at 50 ÌC for 24 hrs and then were pulverized and passed through a sieve No. 60 before being subjected to sample preparation and HPLC analysis. Extraction A 2 kg dried powder of AP leaves was extracted by percolation with ethanol. The alcoholic filtrate was concentrated under reduced pressure to yield a 200 g gummy residue.
93 grapholide (C-2), 4 g (2% yield) of andrographolide (C-3) and 1 g (0.5% yield) of neoandrographolide (C-4) respectively. The combined fractions obtained from 10% to 20% methanol in chloroform, was concentrated under reduced pressure on rotary evaporator and re-chromatograph in a fractional column chromatography packed with silica gel (column size 2.5Ó35 cm). The column was eluted with 10% methanol in chloroform and the fractions of deoxyandrographolide-19-β-Dglucoside (C-5) was collected followed its crystallization in methanol to yield 200 mg (0.1% yield). Identification of the purified diterpenoid lactones Each purified compound was identified by its melting points, UV-spectra, Infrared-spectra, Mass-spectra and TLC (six mobile phase systems; chloroform, chloroform: methanol (9:1), chloroform: methanol (8:1), chloroform: acetone (9:1), chloroform: absolute ethanol (85:15) and chloroform: benzene (1:1) with parallel runs of the standards.
Isolation of diterpenoid lactones A 30 g of gummy residue from the extraction was separated by a silica gel quick column chromatography (25Ó12.5 cm). The column was eluted with chloroform and increase polarity to 20% methanol in chloroform. Each fraction obtained from the quick column was monitored by TLC (Silica gel plate, mobile phase; chloroform: methanol 9:1) and the eluted fractions containing major spots from TLC were combined.
HPLC system for determination of the diterpenoid lactones The standard of dehydroandrographolide (C-2), andrographolide (C-3), neoandrographolide (C-4) and deoxyandrographolide-19-β-D-glucoside (C-5) were identified and quantified by HPLC using reversed phased column of µ-bondapak C18 (30 cmÓ0.39 mm id) and precolumn of LiChrosorb RP-18, 5 µm. The mobile phase of methanol: water (42:58, v/v) flow rate 2 ml/min using detector wavelengths at 255 and 220 nm. The sensitivity was set 0.1 AUFS and the Injection volume of 20 µl.
Crystallization of the isolated compounds The combined fractions eluted with chloroform, 5% methanol and 10% methanol in chloroform were all evaporated separately under reduced pressure to give dark brown syrupy mass. Each syrupy mass was dissolved in methanol, chilled and scratched until crystalline precipitate was formed. The precipitate was collected and further purified by recrystallization from methanol to yield 2 g (1% yield) of dehydroandro-
The standard solutions of C-2, C-3 and C-4 in methanol were prepared in five different concentrations in the range 0.10-0.90 mg/ml, while the standard solution of C-5 was prepared in the range of 0.02-0.2 mg/ml. A 20 µl of each standard solution was injected in duplicate into the column to obtain its chromatogram. The calibration curve was then plotted between peak areas or peak heights against various concentrations of each the standard diterpenoid lactone compound.
94 For reproducibility studied, a 20 µl of each standard solution was subjected to HPLC analysis with five replicates. The concentrations used were as follows: C-2 (0.30 mg/ml), C-3 (0.31 mg/ml), C-4 (0.20 mg/ml) and C-5 (0.08 mg/ml). The obtained data of retention time, peak area and peak height of each injection were then recorded. Determination of the diterpenoid lactones in the leaves of AP Each 250 g of sample powder was extracted with 100 ml methanol in a Soxhletûs extractor for 1.5 hrs. The methanolic extract was concentrated, transferred to a 25 ml volumetric flask and adjusted to the final volume with methanol. For HPLC analysis, a 20 µl of each sample solution was injected replicate under the developed HPLC conditions. The content of each diterpenoid lactone compound was calculated from its individual standard curve.
Results and Discussion The contents of the diterpenoid lactones isolated preparatively from the leaves of AP were as follow: C-2 (1%), C-3 (2%), C-4 (0.5%) and C-5 (0.1%). Each of the isolated diterpenoid lactones compounds was identified based on its physicochemical properties, chromatographic and spectrophotometric aspects (Table 1). The four compounds could be separated simultaneously by our developed HPLC method for using the mixture of methanol: water (48:52) as the mobile phase and µ-bondapak C18 as the stationary phase. Figures 1A and 1B show the typical chromatograms of the sample separation compared with the authentics. The relative retention time (RRt) of each diterpenoid
C. Patarapanich et al. lactone compound appeared to be C-3 (0.22), C-5 (0.45), C-2 (0.75) and C-4 (1.00). The calibration curve of each standard solution showed linearity within its concentration range used. Table 2 shows the calibration parameters of the test samples. For reproducibility, the developed external standard method showed the peak height and peak area of each peak as follows : C-2 (0.89%, 3.76%), C-3 (0.80%, 0.48%), C-4 (1.27%, 4.81%) and C-5 (1.67%, 2.62%) respectively. This results suggested that the peak height gave better reproducibility than the peak area. The data of diterpenoid lactone contents in the leaves of AP planted in various regions in Thailand are summerized in Table 3. All the plant samples were collected within the first or second week of October 1988. The data clearly show that with different location of planting areas, different amount of each diterpenoid lactone was obtained. However the average of the total percentage of diterpenoid lactone in each province was not different significantly although the pattern of diterpenoid lactones appeared to be different. The samples from Suphanburi, Phitsanulok and Chiangmai showed high amount of C-2, but those from Lamphang, Bangkok and Prachinburi showed low C-2 content. The samples from Surattanee, Phuket and Bangkok appeared to have high amount of C-3, while the samples from Sakonnakorn, Prachinburi and Nakhonnayok showed low C-3 content. The samples from Nakhonpanom and Petchabun contain high amount of C-4, while those from Prachinburi and Nakhonnayok showed low C-4 content. Finally the samples from Lampang and Khonkaen showed high amount of C-5, while the samples from Bangkok, Kalasin and Phuket were found to have low C-4 content.
Thai J. Pharm. Sci. 31 (2007) 91-99
(A)
95
(B)
Figure 1 HPLC chromatograms of an artificial mixture of authentic standards (A); and A. paniculata plant extract sample (B), andrographolide (RRt = 0.22), deoxyandrographolide-19-β-D-glucoside (RRt = 0.45), dehydroandrographolide (RRt = 0.75) and neoandrographolide (RRt = 1.00)
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Table 1 Physicochemical properties of the isolated diterpenoid lactone compounds Test item C-2 Physical appearance Melting Range, ÌC (reference MP) TLC: Rf in mobile phase 1. chloroform 2. chloroform: methanol (9:1) 3. chloroform: methanol (8:1) 4. chloroform: acetone (9:1) 5. chloroform: ethanol (85:15) 6. chloroform: benzene (1:1) IR compare to Reference Standard IR of the acetylated derivative Mass spectroscopy (m/e) UV-spectroscopy, λ max, nm.
Diterpenoid lactone compound C-3 C-4
C-5
Needle Colorless 202-203 (203-204)
Plate Colorless 229-230 (230-231)
Needle Colorless 166-167 (167-168)
Needle Colorless 188-190 (187-188)
0.11 0.93 0.92 0.31 0.95 0.95 Conform Conform 332 248, 220
0 0.71 0.76 0.08 0.86 0.95 Conform Conform 350 223
0 0.52 0.54 0 0.65 0.92 Conform Conform 205
0 0.33 0.38 0 0.40 0.90 Conform Conform 205
Table 2 Parameters from the calibration curve of each standard solutions Diterpenoid lactone
Range concentration (mg/ml)
Correlation by Peak height
Correlation by Peak area
C-2 C-3 C-4 C-5
0.10-0.97 0.10-0.42 0.10-0.30 0.10-0.20
0.9990 0.9998 0.9998 0.9991
0.9709 0.9925 0.9886 0.9917
Thai J. Pharm. Sci. 31 (2007) 91-99
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Table 3
Content of diterpenoid lactone compounds from the leaves of AP planted in various regions in Thailand
Region
Percentage weight of diterpenoid lactones found C-5 C-2 C-4
North
South
Central & East
Northeast
Province
C-3
Total
Phitsanulok Lamphang Nakhonsawan Petchabun Chiangmai Average ± SD
2.05 3.83 2.19 3.45 2.27 2.76 ± 0.82
2.28 2.98 2.48 0.61 0.7 1.82 ± 1.07
2.98 0.61 2.15 1.98 2.93 2.13 ± 0.96
1.83 2.07 1.45 2.56 1.56 1.89 ± 0.44
9.14 9.49 8.27 8.59 7.50 8.60 ± 0.77
Prachaubkirikhan Surattanee Phuket Phangnga Ranong Average ± SD
3.41 5.55 5.44 3.72 3.84 4.39 ± 1.02
0.97 0.51 0 0.63 1.45 0.71 ± 0.54
1.39 1.90 1.39 1.16 1.08 1.39 ± 0.32
1.95 1.32 1.18 1.98 1.74 1.63 ± 0.37
7.73 9.29 8.89 7.50 8.11 8.13 ± 0.69
Nakhonnayok Suphanburi Petchaburi Bangkok Chantaburi Prchinburi Average ± SD
2.09 2.54 3.15 5.59 5.77 2.17 3.55 ± 1.69
0.91 1.04 1.04 0 0.86 0.53 0.73 ± 0.40
2.45 3.07 0.91 0.69 1.19 0.78 1.52 ± 1.00
0.81 1.61 1.88 1.47 2.12 0.62 1.42 ± 0.59
6.27 8.26 6.99 7.75 9.94 4.10 7.22 ± 1.97
Sakonnakorn Nakornpanom Surin Khonkaen Kalasin Average ± SD Total (Average) ± SD
1.85 4.13 4.59 2.56 3.09 3.24 ± 1.12 3.49 ± 1.29
1.14 0.73 0.56 2.91 0 1.07 ± 1.11 1.07 ± 0.88
2.49 1.22 1.16 2.78 1.42 1.81 ± 0.76 1.70 ± 0.81
1.22 2.85 1.83 0.98 0.98 1.57 ± 0.80 1.62 ± 0.56
6.69 8.94 8.13 9.23 5.49 7.70 ± 1.58 7.19 ± 2.63
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For seasonal variation, the data of each diterpenoid lactone content in the leaves of AP collected every month in one year-cycle are shown in Table 4. Each diterpenoid lactone content varied significantly during one year time course. The highest C-2 content (7.30 ± 0.48%) was found in April while the lowest (0.61 ± 0.58%) was in December. The highest C-3 content (6.02 ± 0.47%) was found in November, while the lowest (0.82 ± 0.86%) was in February. The highest C-4 content (2.02 ± 0.35, 1.95 ± 0.36%) was found in December-January, while the lowest (0.61 ± 0.58, 0.67 ± 0.53%) was in April-May. The highest C-5 content (3.22 ± 0.22, 3.81 ± 0.39%) was found in February-March, while no C-5 was detected in October- November. However, the highest total diterpenoid lactone contents (11.60, 11.81%) was found in March-April, while the lowest total contents (7.68, 7.80%) was in October-November as compared to the average total content of 9.54 ± 1.47%. From the data obtained, it is suggested that the content of each diterpenoid lactone and the total content in the leaves of AP varied according to the locational and seasonal factors. The Specification of AP in Thai Herbal Pharmacopoeia requires presence of the total lactone content not less than 6.0% w/w, calculated
as the andrographolide. Based on this criteria, it means that the plant sample planted in some period of time or some planting location may meet the specification. Another aspect, at present, the biological activities and the pharmacokinetics properties of each diterpenoid lactone compound still can not be conclusively explained. Therefore, utilization of the AP plant samples or plant extracts with the variation in amount and type of compounds contained may exhibit different resulting activities, although within the total lactone content indicated in the Thai Herbal Pharmacopoeia. Further scientific studies, including the more precised specification of AP need to be demonstrated so that effectively utilization of this Andrographis paniculata Nees. can be achieved.
Conclusion Four diterpene lactones in AP: dehydroandroghapholide (C-2), andrographolide (C-3), neoandrographolide (C-4) and deoxyandrographolide-19-β-D glucoside (C-5) could be simultaneously separated by developed HPLC method for using µ-bondapak C18 and methanol: water (42:58). The relative retention time appeared to be 0.22 (C-3),
Table 4 Content of the diterpenoid lactones from the leaves of AP collected every month in one year-cycle Month C-2 January February March April May June July August September October November December Average ± SD
3.97 ± 0.53 4.18 ± 0.68 5.14 ± 0.55 7.30 ± 0.48 4.43 ± 0.50 5.52 ± 0.64 5.66 ± 0.79 3.58 ± 0.79 1.45 ± 0.55 0.69 ± 0.51 0.84 ± 0.84 0.61 ± 0.58 3.61 ± 2.23
Percentage weight of diterpenoid lactones found C-3 C-4 C-5 1.17 ± 0.60 0.82 ± 0.86 1.01 ± 0.70 1.99 ± 0.71 3.01 ± 0.47 3.13 ± 0.45 3.43 ± 0.41 4.27 ± 0.33 4.86 ± 0.44 5.58 ± 0.51 6.02 ± 0.47 4.43 ± 0.32 3.31 ± 1.78
1.95 ± 0.36 1.51 ± 0.47 1.64 ± 0.43 0.61 ± 0.58 0.67 ± 0.53 0.93 ± 0.76 0.94 ± 0.75 1.27 ± 0.55 1.34 ± 0.53 1.41 ± 0.50 0.94 ± 0.75 2.02 ± 0.35 1.27 ± 0.46
2.03 ± 0.35 3.22 ± 0.22 3.81 ± 0.39 1.91 ± 0.59 0.71 ± 0.99 1.06 ± 0.66 0.88 ± 0.80 0.81 ± 0.87 0.54 ± 0.65 0 0 1.11 ± 1.11 1.34 ± 1.19
Total 9.12 9.73 11.60 11.81 8.81 10.64 10.91 9.93 8.19 7.68 7.80 8.17 9.54 ± 1.47
Thai J. Pharm. Sci. 31 (2007) 91-99 0.45 (C-5), 0.75 (C-2) and 1.00 (C-4). The correlation coefficients (r) of standard curve of all compounds were > 0.97. The method was found to be highly accurate with low RSD < 1.7%. The sample containing the highest amount of C-2, C-4 and C-5 were collected from the northern, whereas the highest amount of C-3 was collected from the southern regions. The highest content of four diterpene lactones in AP was found as follows: C-2 in April; C-3 in November; C-4 in December; C-5 in March. The total diterpene lactone content in AP was found to be highest at 11.81% w/w in April and lowest at 7.68% w/w in October.
Acknowledgement This project has been supported by The National Research Council of Thailand through the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
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