Single-Stage Isolation of Pure Andrographolide From Andrographis Paniculata in a Moderately Polar Solvent By Optimizing Different Parameters.

doi:10.21203/rs.3.rs-2064633/v1

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Single-Stage Isolation of Pure Andrographolide From Andrographis Paniculata in a Moderately Polar Solvent By Optimizing Different Parameters.

https://doi.org/10.21203/rs.3.rs-2064633/v1

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The primary objective of the proposed research work is to extract and enrich the pure Andrographolide (AGL) by varying the polarity index and Hildebrand solubility parameter (δ) of a moderately polar solvent. Both parameters affect the solubility of AGL in different solvents and extraction methods.

AGL was extracted from the whole plant of Andrographis paniculata. Continuous or Direct, Maceration and Soxhlet extraction method used for extraction. Based on the appearance of the crystal in the Direct Soxhlet Ethyl acetate method, their yield was further optimized by using a Randomized response surface (RSM), Quadratic model-based Box-Behnken design (BBD). The HPTLC performed the AGL extraction efficiency, purity, and quantification.

Based on the obtained result, the pure Andrographolide crystal (yield 0.185 g/g) was obtained by Direct Soxhlet extraction. Ethyl acetate is used as a solvent. R_f exhibited in the TLC chromatogram was 0.28. FT-IR spectra exhibited characteristics peak similar to the standard AGL, and ¹H-NMR, ¹³C NMR elucidated crystal structure. DEPT − 90/135 spectra depicted that extracted crystal was pure AGL.

It is one of the novel approaches to isolating pure AGL crystal in a moderately polar solvent (Ethyl acetate) by elevating the Hildebrand solubility parameter despite using polar solvents.

AGL crystal was obtained from Direct Soxhlet extraction by Ethyl acetate. It is a single-step and more economical method. It can be easily transformed into a pilot or industrial setup to extract Pure AGL.

Analytical Chemistry

Biochemical Research Methods

Drug Discovery, Design, & Development

Andrographis paniculata

Andrographolide

Soxhlet

NMR

HPTLC

Crystal

Andrographolide (AGL) is an active phytoconstituent of Andrographis paniculata. Chemically it is a diterpenoid lactone ring. ¹. Due to AGL's variable solubility and polarity in different solvents, it can not be purely isolated by reported methods. ^2,3. Microwave, ultrasonic, and supercritical fluid-assisted extraction techniques were used for AGL's high-yield extraction. However, further chromatographic separation (Column, Flash Chromatography) was needed to get pure AGL. Although these are modern, sophisticated techniques, they require multiple stages to get a pure compound. In the present scenario, the essential key factor in the field of herbal technology is the extraction of a single compound in a single-step process, rather than the high-yield extraction of multiple components followed by the numerous separation techniques used to get pure compound. Extraction techniques can quickly isolate a single phytoconstituent by simulating the solvent's polarity index value or Hildebrand solubility parameter (δ) to the respective bioactive compounds.⁴.

Solubility plays a vital role in the extraction of the compound. Hildebrand's solubility parameter (δ) states that the solubility of any bioactive compound in any solvent is based on its polarity or solubility parameter.⁵.

The square root of the cohesive energy density is expressed as the Hildebrand solubility parameter (δ), which describes the ratio of the heat of vaporization to the molar volume of the compound at a definite temperature. ⁶.

This research comprises different approaches to enhance AGL extraction. To improve the extraction of pure AGL, various solvents (Hexane, Pet. Ether, Toluene, Ethyl acetate, and Methanol) and methods (Direct/Continuous; Cold Maceration/Soxhlet) were used; among these best solvent (Ethyl acetate) and method (Direct Soxhlet) were selected based on obtained AGL crystal. The parameter of the selected method was optimized to increase % the yield of Pure AGL.

Variation of temperature and nature of solvent influence the extraction of AGL ⁷. In the modern era, along with extraction, the isolation of a single bioactive compound is a significant concern. Various novel extraction techniques were developed, like microwave and Supercritical fluid extraction. However, this method is not cost-effective regarding technology transfer to industrial setups concerning conventional methods. The research is based on developing a single-state cost-effective extraction method for a single bioactive compound—enhancing the Hildebrand solubility parameter of a moderately polar solvent. At elevated temperatures, it plays a significant role in the solubility of AGL. At higher heat of vaporization, the equilibrium establishes between the δ value of solvent and solute; as a result, the solubility of solute increases.⁸. However, at ambient temperature, the δ values of solute and solvents increase, which subsequently affects the crystal formation of solute.

Abbreviations: AGL, Andrographolide, TLC. Thin Layer Chromatography; HPTLC, High-Performance Thin Layer Chromatography; UV, Ultra Violet; FT-IR, Fourier Transform Infra-Red; NMR, Nuclear Magnetic Resonance;

2.1 Material

Plant Material: Andrographis paniculata was collected from the local geographical area Pattambi, Kerala, and authenticated (ACCESSION No.205) Whole plant was dried at ambient temperature for a day and then kept in a hot-air oven below 60 °C. It grinds to make 40 mesh powdered and appropriately stored for further use: Hexane, Pet. Ether, Toluene, Ethyl Acetate, and Methanol were purchased from Merck, India. Andrographolide standards were purchased from Nature remedies, Bangalore, India (Purity > 95% by HPLC), and Pre-coated silica gel plate 60 (F₂₅₄) was purchased from Merck, India.

2.1 Extraction and Isolation

Extraction was performed with slight modification in the method reported by Rajani et al., The whole dried plant of A. Paniculata converted into the coarse powder were subjected to extraction by two different methods(Table 01)(Direct and Continuous; Cold Maceration and Soxhlet extraction) ⁹. In Continuous Cold Maceration and Soxhlet, different solvents (Hexane, petroleum ether,

Toluene, Ethyl acetate, and methanol) were used in increasing polarity order [¹⁰],[¹¹],[¹²]. However, in Direct Cold Maceration and Soxhlet extraction, Ethyl acetate and Methanol were used as a solvent. Based on crystal yield, Direct Soxhlet extraction in Ethyl acetate was selected, and their extraction parameter (solute: solvent) proportion, temperature, and extraction time were optimized using expert software version 13.

Randomized response surface (RSM), Quadratic model-based Box-Behnken design (BBD) were used for the optimization of independent variables; Proportion of solvent for 1 part of solute (50 g), Temperature (C), and Time (Minute) at three coded level (-1,) low, (0) moderate and (+1) high level, to get a maximum response in terms of % Yield (w/w) and % Andrographolide (w/w) (Table 02). Seventeen experimental runs with five central points were performed (Supplementary information 1).

. A relation between the independent variable and dependent response is expressed by polynomial Eq (1)

Y1/Y2=β0 + β1 A + β2 B + β3 C + β12 AB + β13 AC + β23 BC + β11 A² + β22 B² + β33 C²---- (01)

Where Y1 (% Yield of extract) and Y2 (% AGL), β0 represent constant β1, β2 and β3 are linear coefficients, β12, β13, and β23 are interaction coefficients, and β11, β22, and β33 are quadratic coefficients. A, B and C are coded values for (Solvent part), Extraction temperature (^oC), and Extraction time (min.), respectively.

2.2 Identification of Andrographolide by HPTLC

0.7 mg of Standard Andrographolide and different extracts were accurately weighed and dissolved in 1 mL of methanol. Pre-coated silica gel plate 60 (F ₂₅₄, 10×10 cm) was used as the Stationary phase, and Toluene: Ethyl acetate: Formic acid (5:4.5:0.5 v/v) was used as the mobile phase.

The applicator spotted a fixed concentration strength (42 µg) or 60µLspot of standard along with different extracts and crystals (Camag, Linomat V, Anchrome, Switzerland). Abbreviation of different extracts; Continuous Soxhlet extraction hexane (SH), Continuous Soxhlet extraction Pet ether (SPE), Continuous Soxhlet extraction toluene (ST), Continuous Soxhlet extraction Ethyl acetate (SEA), Continuous Soxhlet extraction Methanol (SM), Continuous Cold Maceration Hexane (CHE), Continuous Cold Maceration Pet. Ether (CPE), Continuous Cold Maceration toluene (CT), Continuous Cold Maceration Ethyl acetate (CEA), and Continuous Cold Maceration Methanol (CM). The direct extract obtained from Methanol and Ethyl acetate by Cold Maceration is denoted as S1 and S 2, and the Direct extract obtained from Methanol and Ethyl acetate by Soxhlet extraction is denoted as S 3 and S 4. Crystal obtained from Soxhlet Ethyl acetate extract denoted as S 5.

The TLC plate was developed by placing the plate in a saturated glass chamber (10×10 cm, Cammag, Anchrome, Switzerland). Glass Chamber was saturated with the mobile phase. The plate was dried for 1 hr at 105 C, and the Camag TLC visualizer captured the HPTLC fingerprint profiler under UV at 254 nm and 366 nm.

2.3 Quantification of AGL by HPTLC

The developed TLC plate was scanned (Camag TLC Scanner IV, India) at a wavelength of 254 nm. Detection and data evaluation studies can be performed by Wincats, an integrated Software 4.02. The AUC at the respective R_f was determined for the quantitative investigation of the pure compound in the extract.

2.4 Determination of % Purity

The % purity of each tested extraction technique was checked. Obtained dry matter content was expressed in % and calculated from the equation below in Eq. 01

2.5 Identification of Andrographolide by FT-IR

FT-IR spectrophotometer (Agilent, CARY630, Malaysia) was used to identify the functional group. A small quantity of standard AGL and different dried extract and crystals was placed into the sample holder, and run the spectra in the range of 4000-600 cm^-1 and determined the Intensity of % transmittance. Characteristics frequency of standard and test samples were compared to identify compounds.

2.6 Structure elucidation of Andrographolide crystal by NMR

¹H-NMR, ¹³C NMR, and Distortionless enhancement by polarization transfer (DEPT-90/135) NMR spectrum of AGL crystal sample (S5) recorded on Bruker AVANCE Ascend 400 (400MHz FT-NMR) Spectroscopy in Chemical Science Department of IISER-Berhampur, India, and CD₃OD used as a solvent. Structure elucidation was carried out by interpreting the obtained spectrum.

3.1 Extraction of AGL

Based on the selected experimental design, different independent variables, like the proportion of solvent (Ethyl acetate) to 1 part of solute (50 g) coarse powder, was optimized at three level, given in Table 01. Effect of each coefficient on response expressed in terms of positive and negative correlation coefficient given in Eq. 02 and 03. The positive value of the interaction coefficient indicates that extraction and AGL yield was increased on increasing their value; a negative value indicates that the response value decreases on increasing the variable values. The effect of each interaction coefficient on response can be expressed by the response surface plot given in Fig. 01. The optimized value for all the factors was predicted at 4.34 part of Ethyl acetate, 117 ^oC temperature, and 74 min. The observed response % extract yield and % of pure AGL crystal w/w were obtained at 19.049 % and 0.215 g/g, respectively. It was nearly equal to the predicted value (Extraction Yield 19.054 % and % Andrographolide 0.225). It seemed that the proposed model is significant for characteristics response.

3.2 Identification of Andrographolide by HPTLC

Fig. 02 depicted that the spot of standard appeared at R_f 0.28. Spot at similar R_f appeared in different extracts at 254 and 366 nm. Methanol and Ethyl acetate extract derived from various methods exhibited spot at R_f0.28 on 254and 366 nm.

3.3 Quantification of AGL by HPTLC

Fig. 03 Exhibited HPTLC chromatogram of standard crystal (S5) and extracted compound obtained from different extraction methods, which appeared at the R_f value of 0.28. The chromatogram exhibited that Sample S5 (AGL crystal) has a maximum AUC at R_f 0.28.

3.4 Determination of % Purity

Fig. 04, a single spot at Rf 0.28, exhibited that the purest AGL crystal was obtained from Direct Soxhlet extraction in Ethyl acetate. However, Fig. 02 exhibited that extract of other methods shows multiple spots in The TLC plate, revealing that it contains multiple compounds along with AGL, so it required further separation to get pure compound.

3.5 Identification of Andrographolide by FT-IR

FT-IR spectra exhibited that characteristics peaks of crystal Sample S5 appear at 3387 cm^-1 (-OH stretching), 2925 cm^-1 (sp³ C-H Stretching), 2846 cm^-1 (C-H stretching), 1717 cm^-1 (-COOR stretching), and 1669 cm^-1(C=C Stretching) similar kind of peaks were observed in AGL standard. FT-IR spectra of other extracts have low-intensity characteristics peaks shown in Fig. 05.

3.6 Structure elucidation of Andrographolide crystal by NMR

¹H-NMR spectra Fig. 06 and 07 exhibited characteristics signals at

δ 6.85 (t, 1H), 5.03 (d, J = 5.8 Hz, 1H), 4.91 (s, 1H), 4.69 (s, 1H), 4.48 (dd, J = 10.2, 6.1 Hz, 1H), 4.20 – 4.09 (m, 2H), 3.48 – 3.35 (m, 2H), 2.71 – 2.52 (m, 2H), 2.49 – 2.38 (m, 1H), 2.04 (td, J = 13.0, 8.1 Hz, 1H), 1.97 – 1.91 (m, 1H), 1.84 (ddd, J = 18.5, 13.0, 3.6 Hz, 4H), 1.45 – 1.27 (m, 3H), 1.25 (d, J = 8.4 Hz, 3H), 0.77 (s, 3H).

¹³C NMR Fig. 08 and 09 spectra exhibited signals at δ 171.24, 147.93, 147.39, 128.41, 107.81, 79.53, 74.73, 65.26, 63.58, 56.01, 54.94, 48.23, 48.02, 47.80, 47.59, 47.38, 47.16, 46.95, 42.29, 38.57, 37.58, 36.74, 27.64, 24.31, 23.81, 21.97, 14.13.

DEPT-90/135 NMR

The obtained crystal sample is further analyzed the DEPT-90/135 NMR in Fig. 10, and 11 significant differences are observed in DEPT-135, and DEPT-90, respectively; the spectrum of DEPT-135 shows phases methyl positively (-CH₃), methine (-CH) Carbons and negatively phases of methylene (-CH₂) carbons, further quaternary carbon (C) no signals are observed [¹³,¹⁴]. Both DEPT-90 and 135 signals and chemical shifts values are given in Table: 03, similar to previously reported data of Qi Zhen D et al. l ¹⁵ and Chen ZG at all ¹⁶.

4.1 Extraction of AGL

The polarity of the solvent is an essential factor affecting plant material extraction (citation). The extraction efficiency of Phytoconstituents increases with increasing solvent polarity (Lapornik et al., 2005; Rafinska et al., 2019) ^17,18. Pure crystal was obtained in Direct Soxhlet extraction by Ethyl acetate compared to other solvents and techniques. Kumoro et al. reported that 0.0174 g/g AGL was obtained after the chromatographic separation of 0.58% of the total extract¹⁹. Mohan et al. also reported 0.16 g/g AGL extraction by microwave followed by the separation technique ²⁰. Similarly, Sharma et al. also reported extraction of AGL in Methanol by using ultrasonication and microwave followed by separation technique to get pure AGL.²¹. However, the optimized method produces more AGL yield (0.215 g/g) in a single-step process with high yield and % purity, along with being cost-effective compared to reported methods so that it can be easily transformed into the industry for bulk production.

As Hildebrand solubility parameter (δ), different solvents like Methanol and Ethyl acetate are used to extract AGL. Cohesive energy can be expressed as the ratio of vaporization energy to molar volume at a fixed temperature. ²² Intermediate polar solvents (Ethyl acetate, dichloromethane, and chloroform) could not significantly extract the diterpenoid lactones at ambient temperature. However, solute dissolved, and its boiling point elevated due to its colligative property. At an elevated boiling point, the heat of vaporization of the solvent also increases. It may increase the value of the Hildebrand solubility parameter (citation). At a certain period, the δ value of Ethyl acetate and AGL become equal; the solubility of AGL increases in Ethyl acetate. However, at ambient temperature, the heat of vaporization and the δ value of Ethyl acetate decreases; as a result, a significant difference occurs in the δ value of Ethyl acetate and AGL, which would lead to causing the formation of AGL crystal.

In Andrographolide extraction, Ethyl acetate, a moderately polar solvent with a high content of -COO group, was more effective than methanol at elevated temperatures. The value of the polarity index of Ethyl acetate increases with temperature and becomes equal to the polarity index of AGL; as a result, the solubility of AGL increases in hot Ethyl acetate, and on cooling, it gets precipitated and gives the AGL crystal in a single step.

At ambient temperature, the δ value of Methanol and AGL is 14.45 and 14.80, respectively. Solute solubility is maximum when the solute and solvent have the same δ. So, AGL remains present in a solubilized form in methanol at ambient and high temperatures. ²³. Crystal was not observed in methanol as compared to Ethyl acetate. The isolation of pure AGL in Methanol requires further separation by chromatography (column, flash), so single-stage isolation cannot be achieved by methanol or solvents with similar δ values.

The Pure AGL crystal was observed in Direct Soxhlet Ethyl acetate compared to Cold Maceration because the δ value of Ethyl acetate matches with the AGL at elevated temperature. The results of percentage purity are shown in Fig. 04. The results indicate that extraction of AGL is effective with Ethyl acetate Soxhlet extraction rather than other solvent and methods.

Analysis of variance study exhibited that (P< 0.0001) and regression coefficient was 0.9959 and 0.9986 for Extraction yield and AGL Yield respectively, it depicted that proposed quadratic model was significant and validated. The statistical analysis of experimental data depicted increasing the temperature, extraction time, and solvent part. The crystal yield increases as a result of the increased δ value of Ethyl acetate Fig.01 (Supplementary Fig.01). To extract AGL crystal from 1 part of crude drug, approximately 8.6 part of the solvent, 124 C temperature and around 1 hr time was required in Direct Soxhlet extraction (Supplementary Table 02).

4.2 Identification of Andrographolide by HPTLC

Fig. 02 exhibited that AGL was not extracted in non-polar solvents due to the considerable variation in the polarity index and the δ value. It is extracted in moderate polar (Ethyl acetate) and polar solvent (Methanol), which gives a clear spot similar to the R_f (0.28) value of standard AGL.

4.3 Quantification of AGL by HPTLC

Based on observed chromatogram Fig. 03 and 04 exhibited that rich fragment of AGL present in Soxhlet Ethyl acetate crystal (S5) > Direct Cold Maceration Methanol (S1) > Continuous Cold Maceration Methanol (CCM)> Direct Cold Maceration Ethyl acetate (S2) > Continuous Cold Maceration Ethyl acetate (CEA) > Continuous Soxhlet extraction Methanol (SM)> Continuous Soxhlet extraction Ethyl acetate (SEA)> Direct Soxhlet extraction Ethyl acetate (S4) > Direct Soxhlet extraction Methanol (S3)> Continuous Cold Maceration Toluene (CT). The observed result found that in S5 pure AGL crystal was obtained, and in other techniques, AGL was present in solubilized form with other polar constituents.

The polar solvent like methanol gives better AGL yield at ambient temperature in the Cold Maceration method; however, moderate polar solvents like Ethyl acetate give pure crystal at elevated temperature in Soxhlet extraction, and non-polar solvents like hexane, pet. Ether and toluene were not suitable for AGL extraction.

In Cold Maceration, a high polar solvent extract a high amount of AGL due to a similar δ value, but further separation is required to get a pure compound. Ethyl acetate extract obtained from Soxhlet and Cold Maceration was examined. AGL crystal only appeared in Soxhlet Ethyl acetate extract; it inferred that at elevated temperature, the solubility of AGL increased in Ethyl acetate, and on cooling, it gets precipitated and appeared in the form of crystal.

4.4 Determination of % Purity

Fig. 04 exhibited that the purest AGL crystal obtained from Direct Soxhlet extraction in Ethyl acetate ascribes that polarity of Ethyl acetate increases at elevated temperature and reaches the similar polarity index of AGL and extract pure AGL in the form of crystal it reveals that extraction efficiency highly affected by temperature ²⁴. In another method, extract consists of numerous compounds along with AGL. The percentage of AGL present in the different extracts was given in Fig. 04; it depicted that variation in polarity by the effect of temperature may affect the extraction of AGL. Quantification of amorphous extract obtained from different solvents depicted that Methanol extract has the rich fragment of AGL as compared to Ethyl acetate and another solvent but required further purification for isolation of pure AGL.

4.5 Identification of AGL by FT-IR

The peak of crystal (S5) appears at 3387 cm^-1 (-OH stretching), 2925 cm^-1 (sp³ C-H Stretching), 2846 cm^-1 (C-H stretching), 1717 cm^-1 (-COOR stretching), and 1669 cm^-1(C=C Stretching) shown in Fig. 05. All the observed peaks of crystal (S5/ AGL-DSEAC) exactly matched with standard AGL as compare to other extracts. Similar peaks were reported by Mishra et al. ²⁵ for the identification of AGL.

Solvent polarity may affect the Shifting of FT-IR peak of organic compounds due to solvent interactions, called solvent effects. ^26,27. Apart from AGL-DSEAC other extracts showing shifting of the signal as compared to the standard peak as a result of solvent interaction and the influence of temperature ²⁸The formation of the hydrogen bond with solvents shifted in the FT-IR spectra peak. The AGL-DCMM ester group peak appeared at 1650 cm^-1;it sifted to a lower frequency exhibiting a red shift in the spectra. It might occur due to solvent hydrogen bond interaction with the carbonyl group of AGL.

In AGL-DSEA, -COOR stretching peak appeared at 1736 cm^-1it shifted to a higher frequency concerning AGL-STD as a result of the blue shift, whereas in AGL-DCMEA, same peaks were observed at 1702 cm^- shifted to a lower frequency in concert with the standard peak as a result of red-shift, it might occur due to the solvent interaction with AGL under the influence of temperature. The shifting of frequency is directly associated with the effect of temperature. Stretching frequency increases with the elevation of temperature ²⁹. Fig.05 exhibited that Methanol and Ethyl acetate extract have characteristics peak of AGL and crystal obtained in Ethyl acetate was precisely match with the AGL-STD peak it depicted that crystal was chemically pure AGL which further confirmed by other sophisticated analytical techniques.

4.6 Structure elucidation of Andrographolide crystal by NMR

Fig. 06 exhibited that AGL is a bicyclic diterpenoid lactone with three Hydroxyl groups at C-3, C-14, and C-19. Proton present at this respective carbon are secondary, allylic, and primary, respectively ³⁰.¹H NMR spectra Fig. 07 NMR spectra exhibited that S5 three proton signals of alcoholic hydrogen did not appear in ^{the 1}H-NMR spectrum; it inferred that alcoholic protons interchanged with the deuterium isotopes of deuterated Methanol (CD₃OD) used as a solvent. CD₃OD of deuterium isotopes are easily interchanged with active hydrogen of alcohol protons; as a result, protons diminished in the ¹H-NMR spectrum of AGL ³¹.

The S5 ¹H NMR spectrum depicted 27 Hydrogen and the presence of methyl groups H-18 at δ 1.27-1.45 (3H) and H-20 at δ 0.77 (3H), respectively. Fig. 07 exhibited that signal of methylene group at aliphatic chain H-11at δ 2.52-2.71 (2H) and in lactone ring H-15 at δ 4.09-4.20 (2H), and in cyclic hexane ring H-1 at δ 1.27-1.84 (2H), H-2 δ 1.84 (2H), H-6 δ 1.45-1.84 (2H) and H-7 δ 2.08-2.45 (2H) respectively. Hydrogen at tertiary carbon appeared at H-5 at δ 1.36 (1H), H-9 at δ 1.93 (1H). Another characteristic signal of AGL can be confined by the appearance of two double bonds, hydrogen signal H-12 at δ 6.85 (1H) and H-17 δ 4.69 (2H), respectively. The signal appeared at H-3 δ 4.48 (1H), H-14 δ 5.03 (1H), and H-19 δ 3.35-3.48 (2H), depicted that proton present at secondary, allylic, and primary alcoholic carbon shown in Fig. 07 Similar investigation was reported by Levita, Jutti, et al^{. 32.}

The triplet at H-12 δ 6.85 confirms the double bond hydrogen. The diastereotopic methylene protons at H-15a and 15b δ 4.09-4.20 (2H) data are similar to the 1H-NMR of andrographolide ³³. The appearance of the signal at characteristic δ value ascribes that obtained crystal was Andrographolide further ¹³C and DEPT 90/135 analysis also confirmed their structure.

Figures 08 and 09 depict that AGL is a bicyclic diterpenoid lactone with 20 Carbons. The S5 was further characterized by ¹³C NMR spectroscopy, and CD₃OD was used as a solvent; in this study, the Chemical shift values of S5 (Table 02) found the lactone Carbonyl (C-16) and two methyl groups (C-18, 20) at δ 171.24 and δ 21.97, δ 14.13 respectively [³⁴]. The Characteristic Exo double bond (C-17) at δ 107.81, lactone ring attached double bond (C-12, C-13) at δ 147.39, 128.41, and an endocyclic double bond at (C-8) δ 147.93. The hydroxyl group attached carbons (C-3, C-14, and C-19) signals are observed at δ 79.53, 65.26, and 63.58 secondary, lactone ring and methylene carbons observed, respectively. Further, the cyclohexane ring of methylene carbons (C-1, C-2, C-6, and C-7) signals at δ 37.58, 27.64, 24.31, and 36.74 appeared in ¹³C NMR Spectrum. The methylene carbon (C-15) present in the lactone ring signal shows a filed value at δ 74.73 due to the deshielding effect of the ring [³⁵].

The S5 ¹³C NMR spectroscopy data of tertiary (C-5) at δ 54.94 and quaternary carbons (C-4, C-10) at δ 42.29, 38.57 was confirmed. All the observed values match the value proposed in Fig. 09; the obtained crystal was pure AGL.

DEPT-90/135 NMR

Fig 10 and 11 exhibited shifting of a peak in positive or negative phase complies with reported value for standard given in Table 03 and matches with the value predicted by Chem Draw exhibited in Fig. 06 and 09 it refers that obtained crystal was AGL.

Solute: solvent ratio, extraction temperature, and time play a significant role in acquiring the optimum δ value of Ethyl acetate at which AGL dissolved and crystallized when solvent reached ambient temperature. The new finding of the proposed work is to optimize the extraction parameters for mild polar solvent (Ethyl acetate). This finding concludes that a high yield of pure AGL was extracted in a single stage in moderate polar solvent compared to previous reported polar solvents (methanol, ethanol). This optimized method can extract pure AGL crystal in a moderately polar solvent by minimizing the polarity index and Hildebrand solubility parameter difference of AGL and Ethyl acetate. This method is more economical for pure crystal isolation, and in other extraction methods, multi-components were obtained along with AGL. The AGL extraction percentage varies with extraction parameters (polarity of solvents, temperature) and methods. Ethyl acetate is a good choice of solvent in the Soxhlet method and Methanol for Maceration method to extract AGL.

Acknowledgment: Authors Acknowledge Dr.D.Sudhakar Director NARIP and Dr. Rabinarayan Acharya DG CCRAS for the guidance provided for completing the paper successfully.

Ethical Approval: Not applicable

Consent to Participate: Not applicable

Consent to Publish: Not applicable

Competing Interests: Authors do not have any competing interests.

Funding: Not applicable

Conflict of interest: Authors do not have any conflict of interest.

Authors’ contributions: Rahul Maurya, B Thirupataiah, Lakshminarayana Misro, R Thulasi, V Rajeesh, and K Rohit involves designing hypotheses, experiments, and preparation of the manuscript. Dr.D.Sudhakar, Director NARIP, and Dr. Ravinarayan Acharya, DG CCRAS, provide institutional support.

Availability of data and materials: The data supporting the finding of this study are available from the corresponding author upon reasonable request.

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Single-Stage Isolation of Pure Andrographolide From Andrographis Paniculata in a Moderately Polar Solvent By Optimizing Different Parameters.

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