INTRODUCTION:

Since thousands of years ago, natural products have been used traditionally to treat various diseases, maintain fitness and increase endurance. Natural products provide a variety of phytochemical compounds that are active against different biological functions¹.

The presence of complex bioactive components in plant-based natural products plays a very important role in the design and development of new drugs, not only that but medicinal plants also have an important role as commodities in international trade. Metabolites, both primary and secondary, make up phytochemical components. Numerous biological effects of secondary metabolite molecules, including antioxidant, antibacterial, antifungal, anticancer, anti-inflammatory, and antidiabetic properties, have been demonstrated. Plants produce many chemicals known as secondary metabolites, such as alkaloids, flavonoids, phenolics, saponins, and terpenoids².

Ruellia tuberosa L. is a weed plant that is commonly found in damp, shaded settings in tropical nations and has long been used as a medicinal herb. As a member of the Acanthaceae family, Ruellia tuberosa L., often referred to as the pletekan or popping pod plant, is extensively found throughout Central America, Africa, and South and Southeast Asia.Various parts of this plant have been utilized in ethnobotany to treat kidney problems, antidotes, rheumatism, hemorrhoids, and diabetes, lung disorders to sexually transmitted diseases. The tuberous roots of Ruellia tuberosa L. function to enable the plant to survive during the dry season, even its ripe seed capsules can burst when water drops fall on the capsules causing the seeds to spread far from the plant. The tubers are reported to contain n-alkanes, triterpenes, phytosterols and lupeols. In addition, compounds such as alkaloids, steroids, terpenoids and saponins have been isolated from the roots of Ruellia tuberosa. Ruellia tuberosa L. is rich in secondary metabolite compounds such as glycosides, flavonoids, alkaloids, terpenoids, and steroids. Over the past 10 years several studies related to extracts from Ruellia tuberosa L. leaves have been reported. The methanol extract of Ruellia tuberosa L. leaves is reported to contain phytochemical compounds such as organic acids, alkaloids, aldehydes, flavonoids, glycosides, ketones, saponins, tannins and terpenoids which show antimicrobial, antioxidant, analgesic, diuretic, anti-inflammatory, antinociceptive and anticancer activities^3-6.

The study reported by⁷ who conducted an analysis of the total content of alkaloid, flavonoid, tannin and phenolic compounds and proved the pharmacological activity as an anticholinesterase and antioxidant of chloroform extracts from leaves, stems and roots of Ruellia tuberosa L. The study's findings revealed that anticholinesterase activity was better at a dose of 20 µg/mL compared to antioxidant activity, it is suspected because the content of polyphenol compounds has a lower concentration than alkaloid and flavonoid compounds. Hydroethanol extract of Ruellia tuberosa L. roots provides activity as a DPPH radical scavenger at a very strong concentration and reduces malondialdehyde (MDA) levels which play a role in causing oxidative stress^8,9. Ethanol extract of Ruellia tuberosa L. leaves and stems obtained by the soxhletation method has been shown to provide pharmacological activity as an antibacterial and antifungal in Escherichia coli, Staphyllococcus aureus and Candida albicans. Ruellia tuberosa L.'s methanol extract yielded the ethyl acetate fraction, which was found to enhance hepatic glucose absorption and reduce lipid buildup by both enhancing and inhibiting the hepatic insulin signaling and lipogenesis pathways in hepatocytes. Ruellia tuberosa L. is known in traditional medicine to be an antidiabetic; these findings are also supported by scientific research (10). Ruellia tuberosa L. aerial parts ethanol extract shown antinociceptive and anti-inflammatory properties based on a number of well-researched and verified experimental techniques with mice and/or rat models.

In addition to the activities of the extract and active fractions of Ruellia tuberosa L., several compounds isolated from the plant have also been reported. Flavonoid glycoside compounds with very strong activities as DPPH radical scavenging antioxidants and xanthine oxidation inhibitors were isolated from dichlormethane and methanol extracts of Ruellia tuberosa L. leaves⁶. Two phenolic compounds, syringic acid and p-coumaric acid, and one group of flavonoid compounds, cirsimaritin, were all isolated from methanol-water extracts obtained from the stems and leaves of Ruellia tuberosa L¹⁰.

Secondary metabolite compounds in Ruellia tuberosa L. and their contribution to various pharmacological activities provide opportunities to develop and explore other compounds, one of which is found in the leaves. Based on literature searches, during the last 10 years there has been no research examining antioxidant activity and determining the content of metabolites through an in silico approach from compounds identified by the GC-MS method¹¹.

MATERIALS AND METHODS:

Materials:

Ruellia Tuberosa L. leaves was collected in Desember 2023 from Ngasem, Kediri, Indonesia. The specimens were kept in the Laboratory of Pharmaceutical Biology (RT/09/L/2024). Ethanol, methanol, n-hexane, ethyl acetate as solvents (Smart Lab, Indonesia). Folin-ciocalteu, sodium carbonate, aluminum chloride, potassium acetate, gallic acid, and standard quercetin as standard, 2, 2'-Azinobis (3-ethyl-benzothiozoline-6-sulfonic acid) disodium salt, DPPH, TPTZ (Sigma-Aldrich; St. Louis, MO, USA).

Extraction and Fractination Methods.

Dried Ruellia Tuberosa L. leaves was powdered and then 1 kg of powdered material was macerated using 70% ethanol in a ratio of 1:7 over night for 3 days to obtain ethanolic extract Ruellia (EER). The EER was then separated into fractions of n-hexane (HF), ethyl acetate (EAF), and methanol (MF) using a solid-liquid method with solvents ranging from non-polar (n-hexane) to semi-polar (ethyl acetate) to polar (methanol) All fractions were concentrated in a rotary evaporator (Buchi).

Phytochemical Analysis Methods:

Qualitative Phytochemical Screening:

The methods were base on previous research^12-14 with slight modification to evaluate the presence of phenolics, alcaloids, tannins, sapponins, triterpenoids/ steroids and flavonoids group in EER and its fractions.

Quantitative Phytochemical Screening:

a) Determination of Total Phenolic Content (TPC):

The colorimetric approach from Sariwati and Samydurai 15, 16 was modified in order to determine the total phenolic content. Weighed up to 10mg, EER, HF, EAF, and MF were dissolved in up to 10mL of ethanol per batch. After mixing 0.5mL of each of the following: EE, FH, EAF, and MF with 0.4mL of Folin-Ciocalteu reagent, the mixture was incubated for a duration of 4 to 8minutes. Subsequently, the mixture was mixed with 4.0 mL of a sodium carbonate solution (7%), and 10mL of distilled water. After a two-hour incubation period at room temperature, the absorbance at 750nm was measured using a UV-Vis spectrophotometer (Shimadzu UV-1280, Japan). The calibration curve was made using standard solutions of gallic acid at 40, 60, 80, 100, and 120mg/mL. The total phenolic content was determined using the gallic acid equivalent milligrams per gram of sample.

b) Determination of Total Flavonoid Content (TFC):

The colorimetric method (7) was utilized to ascertain the total flavonoid concentration. Weighed up to 10mg, EER HF, EAF, and MF were dissolved in up to 10mL of ethanol p.a. To summarize, 0.5mL of EER, HF, EAF, and MF were mixed with 2.5 mL of distilled water, 0.1 mL of 10% AlCl3, 0.1mL of 1M potassium acetate, and 1.5mL of methanol. With a UV-Vis spectrophotometer (Shimadzu UV-1280, Japan), the absorbance was measured at 415nm following a 30minute incubation period. The results were represented as quercetin equivalents in mg of quercetin per g of sample, with the concentrations of 40, 60, 80, 100, and 120g/mL of quercetin serving as the standard for the calibration curves.

Antioxidant Activity Assay Methods:

a) DPPH Radical Scavenging Assay:

The DPPH assay was carried out according to^15,16 with slight modification. A specific concentration series was created from each sample solution (EER, HF, EAF, and MF). For each series of EER, HF, EAF, and MF, 0.2mL of solution was used to determine the antioxidant activity.and quercetin (standard). The sample was placed in a test tube and, for each concentration, 4.0mL of 0.1 mM DPPH was added. The sample's absorbance was then measured at 517nm precisely 30 seconds after the extract was added (17, 18). The experiment was run in triplicate, and formula no. 1 was used to determine the percentage of scavenging activity:

% Antioxidant activity = (Abs. control – Abs. sample) / Abs. control x 100%…………………………………..1

b) Ferric Reducing Antioxidant Power (FRAP) Assay:

Frap testing is carried out according to the method Benzie and Strain (19) with modification. To create the FRAP solution, 10:1 mixtures of an acetate buffer solution containing 0.1M (PH 3.6), TPTZ (2,4,6-tripydyl-s-triazine) dissolved in 40mM HCl, and FeCl3.6H2O 12mM were used. 10mg of each of the following compounds were diluted in a flask holding 10 mL of methanol: EER, HF, EAF, and MF.

The experiment was run in triplicate, and formula no. 2 was used to determine the percentage of lowering activity:

% Antioxidant activity = (Compound FRAP value/ 100% FeSO₄ FRAP Value) x 100 %…………………..2

c) ABTS Radical Scavenging Assay:

The ABTS assay was performed in accordance with (15,21), modified. After reacting with 7mM ABTS in 140 mM potassium persulfate solution for 12–16 hours in the dark, the ABTS^•+ solution was diluted to have an absorbance of 0.7±0.002. After thoroughly mixing 0.1 mL of the sample solution (EER, HF, EAF, and MF) with 4.9mL of ABTS•+ solution for 10minutes, the absorbance at 736 nm was measured. The experiment was run in triplicate, and formula no. 3 was used to determine the percentage of lowering activity:

% Antioxidant activity = (Abs. control – Abs. sample) / Abs. control x 100%………………………………..3

In Silico Study Methods:

In silico studies in this study used an approach to secondary metabolite compounds contained in the active fraction using the GC-MS method. The materials used in this study consist of the 3D structures of compounds or ligands, specifically 6-Carbomethoxy-Trans-1-Oxaspiro[4.5]Decan-2,8-Dione, Bufa-20,22-dienolide, 14-hydroxy-3-oxo- (5.beta.), 1-Phenyl-2-ethylprop-1-ene (1-3)sultine, Amyl Cinnamic Aldehyde Diethyl Acetal 2, Trans-1-Oxaspiro[4.5]Decan-2,8-Dione, 6-Carbomethoxy, all of which were stored in .sdf format. The protein structure used was human glucokinase (PDB ID: 1V4S) in FASTA format. The protein structure used was human glucokinase (PDB ID: 1V4S) in FASTA format. The tools used in this study include an Acer Swift 3 Laptop (LAPTOP-O4DRN1NB), Intel Evo Core i5-1135G7 @ 2.40GHz 2.42 GHz, 16 GB RAM, running Windows 11, along with software such as PyRx, Autodock Vina, and Discovery Studio 2021. Additionally, the web servers utilized were PubChem (pubchem.ncbi), RSCB (Research Collaboratory for Structural Bioinformatics), and UniProt.org.

Protein and 3D Ligand Preparation:

The purpose of preparing the receptor protein is to remove water molecules, native ligands, and co-receptors from the protein, ensuring that during docking, there is no obstruction in binding to the protein’s active site¹⁷. Both the protein and ligands were prepared using Discovery Studio 2021, and converted into 3D structures using Autodock Vina 4.0. Ligand minimization was performed to optimize binding energy, ensuring greater flexibility of the ligand atoms during the molecular docking phase. This minimization was done using the OpenBabel plugin within PyRx software, resulting in a flexible 3D structure in pdb format. This method is used to simulate the interaction between molecules to obtain a stable binding energy value, enabling the formation of a molecular complex¹⁸.

Data analysis:

Every measurement of TPC and TFC antioxidant activity was done three times. The mean ± SD was used to express the experimental findings. Using IBM ver. 25, a one-way analysis of variance (ANOVA) was performed on the data. For the purpose of identifying mean differences, a post hoc test was employed. At the significance threshold of α< 0.05, it was accepted at a level of 5%.

RESULT:

Phytochemical Analysis:

Qualitative Phytochemical Screening:

The test was based on the color changes after EER, HF, EAF, and MF were reacted with standard reagents to check the presence or absence existence of different phytochemicals in EER, HF, EAF, and MF of Ruellia tuberosa L. The secondary metabolite identified were phenolics, flavonoids, alkaloids, tannins, saponins and terpenoids the results are presented in table 1.

Table 1. Phytochemical screening of Ruellia tuberosa L. leaves

Secondary metabolites	RE	HF	EAF	MF
Phenolic	+	+	+	+
Alcaloids	+	+	-	-
Tannin	+	-	-	+
Sapponins	+	-	-	+
Triterpenoids	-	-	-	-
Flavonoids	+	+	+	+

Note: (+): present; (-): not present

Quantitative Phytochemical Screening:

Only two classes of phytochemicals—phenolic and flavonoid—were subjected to phytochemical quantitative analysis. The total phenolic content and the total flavonoid content were measured (Figure 1). Comparing EAF to other samples, the results showed that it had the greatest total phenolic and flavonoid concentration.

Figure 1. Total phenolics contents/TPCs (mg GAE/g sample) and total flavonoids contents/TFCs (mg QE/g sample) of ethanolic extract Ruellia (EER), hexan fraction (HF), ethyl acetate fraction (EAF), methanol fraction (MF). Values are mean±SD (n = 3), ** are significant (p<0.05); ns = not significant (α>0.05)

Antioxidant Activity:

Antioxidant activity of EER, HF, EAF and MF of Ruellia tuberosa L. leaves against DPPH, FRAP and ABTS radicals can be seen in Figure 2. The result has indicated that the extract and fractions have antioxidant activity against DPPH, FRAP and ABTS radicals. However, HF, EAF and MF have stronger activity than EER, except HF (IC50=139.300 µg/mL) which has lower activity than EER (IC50=131.413 µg/mL) against DPPH radicals.

Figure 2. DPPH radical scavenging activity, FRAP (Ferric Reducing Antioxidant Power) activity, and ABTS scavenging activity of Quercetin as standard, ethanolic extract of Ruellia (EER), n-hexane fraction (HF), ethyl acetate fraction (EAF), methanol fraction (MF) in IC₅₀ (mg/mL). Values are mean±SD (n = 3). *, ** and **** are significant (α<0.05), ns = not significant (α>0.05)

In silico study:

The parameters analyzed in the molecular docking study include the grid box, binding affinity, amino acid residues, types of bonds, hydrogen bonds, and bond distances. In silico molecular docking studies represent an approach utilized to predict interactions between small molecules (ligands) and biological targets (receptors), such as enzymes or proteins, that are involved in specific biochemical processes. The in silico assessment of the antioxidant activity of Ruellia tuberosa L. leaves can be leveraged to forecast the interactions of active compounds with target enzymes or proteins associated with oxidative stress within the body⁹.

This study utilizes receptor 1V4S, which corresponds to human glucokinase. Glucokinase is a cytoplasmic enzyme that plays a crucial role in the phosphorylation of glucose, thereby initiating the utilization and metabolism of glucose in the liver and pancreas. The allosteric properties of glucokinase enable this enzyme to optimally respond to physiological glucose concentrations. Although the molecular mechanisms underlying its function as a glucose sensor remain incompletely elucidated, recent crystallographic studies have provided insights into the conformational changes of the enzyme that facilitate allosteric activation¹⁹.

Figure 3. Structure of 3D Protein Reseptor 1V4S. (left) before preparation; (right) after preparation

DISCUSSION:

The results of a qualitative phytochemical examination revealed the existence of phytochemical elements. These constituents included phenolics, flavonoids, alkaloids, tannins, saponins, and triterpenoids/steroids. These findings revealed that phenolics, flavonoids, alkaloids, tannins, saponins were positively detected in EER, while triterpenoids were not detected. Phenolics and flavonoids were secondary metabolites that were positively detected in all samples. Previous studies also reported that in R. tuberosa extracts in various solvents and different extraction methods resulted in the presence of phenolic compounds, flavonoids, alkaloids, tannins and saponins, except triterpenoids which were not detected(3,20). The group of secondary metabolites identified positively in the sample is influenced by the type of solvent used in the extraction²¹.

Determination of secondary metabolite compound content is focused on two groups, namely phenolics and flavonoids, because these two compounds are widely distributed and abundant in plants in quite large concentrations^22,23. Phenolic group is a large group of secondary metabolites which includes groups of tannin compounds, flavonoids, stilbenes, and phenolic acids²⁴. Phenolic and flavonoid compounds have been shown to provide important contributions to pharmacological activities such as antioxidants, anticancer, anti-inflammatory, antiviral, antifungal and cardioprotective. Determination of TPC and TFC also provides an overview of how much the compound contributes to providing pharmacological activity²³.

The overall phenolic content of the EER and HF, EAF, and MF were varied. EAF (161.18±9.70µg GAE/g) showed the highest TPC and then followed by MF, HF and EER as the lowest TPC (Figure 1). There was no significant difference TPC between MF and EAF. The TPC results in EAF has high values possibly because etil asetat merupakan pelarut yang memiliki sifat semipolar dan banyak menarik berbagai golongan senyawa fenolik. The smallest TPC results were found in EER, the extract contains various secondary metabolites, including phenolic compounds in low levels. While the fractions provide higher TPC due to the selectivity of the solvent used in the separation process. In previous studies⁷ it was also reported that TPC from chloroform extract of R. tuberosa L. leaves gave a result of 0.16±0.01µg GAE/g, but the result was much different from the result in this study (161.18±9.70µg GAE/g). TPC is influenced by the solvent used in the extraction, the chemical structure of phenolics has a hydroxyl group that increases its solubility in polar solvents such as ethanol used in this study, while in previous studies using chloroform which has a lower polarity.

TFC in this study also has the same pattern as the results of TPC determination. EAF is the sample with the highest TFC (224.74±10.16 µg QE/g) followed by MF (186.15±9.13µg QE/g), HF (132.74±3.26µg QE/g) and EER has the lowest TFC (131.46±8.81µg QE/g). As stated above, EER has the lowest TFC concentration because the extract still contains various secondary metabolites with low concentrations, in contrast to the fractions that have been separated based on selective solvents^22,25.

When measuring antioxidant properties, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a radical that is quick, simple, and readily available in the market. It can be used with free radicals to determine whether a substance has the ability to provide hydrogen or scavenge free radicals.(26,27). Quercetin was used as the standard in this study. Our results showed that the HF produced the lowest antioxidant activity (IC₅₀= 139.30±4.35µg/mL). The non-polar character of n-hexane is the cause of the minimal content of compounds with a high number of hydrogen atoms such as flavonoids and phenolics²⁸, this causes a low concentration of DPPH radical inhibition (Figure 2). While the highest activity was EAF (IC₅₀ = 55.51±2.52µg/mL) followed by MF (IC₅₀ = 68.33±1.12 µg/mL), and EER (IC₅₀ = 131.41±3.58µg/mL). EAF provides the strongest scavenging activity against DPPH radicals because the semipolar character of ethyl acetate attracts many groups of polyphenol compounds such as tannins, flavonoids, to glycosides that have semi-polar to polar properties, so it can be concluded that EAF contains compounds that actively capture DPPH radicals. Based on previous research²⁹, methanol extract and its fractions tested against DPPH radicals gave similar results to this study. Previous studies also reported that ethyl acetate and chloroform fractions were the strongest fractions against DPPH radicals, while methanol extract and water fractions gave the lowest inhibition³⁰.

In this study, the FRAP assay was used to determine the antioxidant strength in reducing ferric ions to ferrous ions. One electron from the extract metabolite is transferred to the ferric ion³¹. In this study, the reduction capacity of the samples was EAF> MF> HF> EER (Figure 2), these results indicate that EAF contains active ingredients that can transfer electrons to the Fe³⁺TPTZ complex which is considered to represent the oxidant in the body to Fe²⁺TPTZ. The capacity of EAF (IC₅₀ = 53.55±2.89µg/mL) is significantly different from the quercetin control (IC₅₀= 9.40±0.41µg/mL), quercetin is a single compound that has electrons to reduce Fe3+ to Fe2+. Meanwhile, EAF still contains other compounds that do not have the ability to reduce.

The result of ABTS assay provide the same pattern as the results of the FRAP assay (Figure 2), with each inhibition concentration EAF (IC₅₀=42.77±1.13 µg/mL)>MF (IC₅₀=53.31±2.63 µg/mL)>HF (IC₅₀=108.99±4.67µg/mL)>EER (IC₅₀=112.52±3.44 µg/mL). In the ABTS^.+ assay, electron transfer occurs which will reach the end point where different antioxidant compounds donate one or two electrons to reduce radical cations³².

Antioxidant potential of plants is frequently linked to their flavonoid and phenolic component contents³³. Because phenolic compounds have antioxidative characteristics, they have been reported to have antioxidant activity. In addition to being potential chelators, phenolic compounds also function as hydrogen donors, reducing agents, and singlet oxygen absorbers. Because flavonoid compounds may donate hydrogen atoms to free radicals, they can neutralize them and act as antioxidants. The antioxidant action of flavonoids is attributed to the presence of phenolic hydroxy groups and phenolic groups in their molecular structure.

The molecular docking study (table 2) indicate that ligands with high binding affinity, such as Bufa-20, 22-dienolide, may possess stronger antioxidant activity. However, the lack of interaction similarities with residues involved in the native ligand raises questions regarding its biological relevance. Despite this compound binding strongly to the receptor, if the ligand-receptor interaction mechanisms differ significantly, the resultant effects may not align with expectations. Conversely, Trans-1-Oxaspiro[4.5]Decan-2,8-Dione, 6-Carbomethoxy offers a different perspective. Although it has a slightly lower binding affinity, its high interaction similarity with the native ligand provides hope that this compound may operate through similar mechanisms, potentially yielding improved therapeutic efficacy as an antioxidant. Additionally, 6-Carbomethoxy-Trans-1-Oxaspiro[4.5]Decan-2,8-Dione shows promise despite its low interaction similarity. This suggests that structural modifications may be necessary to enhance its interaction similarity with the receptor, thereby improving its biological efficacy^34,35. Compounds with low binding affinities, such as 1-Phenyl-2-ethylprop-1-ene (1-3)sultine and Amyl Cinnamic Aldehyde Diethyl Acetal 2, appear to have reduced potential as antioxidant agents. Their low binding affinities and minimal interactions with crucial receptor residues indicate that these ligands may exhibit weak binding stability and may not be effective in the desired biological applications

CONCLUSION:

Ethyl acetate fraction is an active fraction of Ethanolic Extract Ruellia which has antioxidant activity and capacity through the mechanism of free radical scavenging (electron transfer and hydrogen atom donor) and reducing Fe³⁺ to Fe^2+. The antioxidant potential of EAF is believed to be the contribution of bioactive compounds that are rich in electrons and hydrogen atoms, so that EAF also has the highest TPC and TFC compared to other samples. Ethyl acetate fraction is an active fraction of Ethanolic Extract Ruellia which has antioxidant activity and capacity through the mechanism of free radical scavenging (electron transfer and hydrogen atom donor) and reducing Fe³⁺ to Fe^2+. The antioxidant potential of EAF is believed to be the contribution of bioactive compounds that are rich in electrons and hydrogen atoms, so that EAF also has the highest TPC and TFC compared to other samples. 6-Carbomethoxy-Trans-1-Oxaspiro[4.5]Decan-2,8-Dione presents opportunities for further development, but may require structural modifications to enhance its efficacy. A combined approach involving docking analysis and experimental validation, both in vitro and in vivo, is crucial for determining the true biological potential of these ligands as effective antioxidant agents.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank to Ministry of Education, Culture, Research and Technology. Directorate General of Higher Education, Research and Technology through research grants for novice lecturer research schemes for the 2024 budget year with contract number 087/SP2H/PT/LL7/2024. The authors also thank to Institut Ilmu Kesehatan Bhakti Wiyata Kediri for laboratory facilities and infrastructure.

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Received on 29.09.2024 Revised on 01.02.2025

Accepted on 03.04.2025 Published on 05.09.2025

Available online from September 08, 2025

Research J. Pharmacy and Technology. 2025;18(9):4497-4504.

DOI: 10.52711/0974-360X.2025.00645

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Ligan	Binding Affinity (Kkal/mol)	Amino Acid Residue	Total Amino Acid Residues Compared to Nativ Ligand	Percentage of Amino Acid Similarities (%)
2-Amino-4-Fluoro-5-[(1-Methyl-1h-Imidazol-2-Yl)Sulfanyl]-N-(1,3-Thiazol-2-Yl)Benzamide (Nativ Ligand)	-7,1	Conventional Hydrogen Bond: Lys 169, Asp 78, Ser 151, Ser 445, Thr 228; Carbon Hydrogen Bond: Gly 81 Ser 445 Pi-cation: Asp 205, Arg 85 Pi-anion: Asp 205, Arg 85	11	100
6-Carbomethoxy-Trans-1-Oxaspiro[4.5]Decan-2,8-Dione	-6,8	Conventional Hydrogen Bond: Gly 229, Thr 228, Ser 411, Ser 151	2	18,1
Bufa-20,22-dienolide, 14-hydroxy-3-oxo-, (5.beta.)	-7,9	Conventional Hydrogen Bond:Ser 280, Arg 327 Pi-anion: Glu 272 Pi-Alkyl: Arg 308	0	0
1-Phenyl-2-ethylprop-1-ene (1-3)sultine	-5,9	Attractive Charge : Asp158 Pi-Donor Hydrogen Bond : Agr63 Alkyl & Pi-Alkyl : Lys459, Val62, Val455, ile159, Pro66, Ala456	0	0
Amyl Cinnamic Aldehyde Diethyl Acetal 2	-5,9	Van der Waals : Ala456, Arg63, Ile159, Tyr61, Asp158 Alkyl & Pi-Alkyl : Val452, Val455, Val62, Pro66, Lys459	0	0
Trans-1-Oxaspiro[4.5]Decan-2,8-Dion, 6-Carbomethoxy	-6,0	Van der Waals :Gly229, Thr228, Gly227, Ile225, Ser411, Gry410, Asp78, Asp205, Gly80, Ser151, Gly81 Conventional Hydrogen Bond : Lys169, Ser445, Arg85 Carbon Hydogen Bond : Asp409	7	63,6