# In Vitro Anti-Inflammatory, Anticancer (MCF-7, 3T3, and HeLa Cell Lines), and Brine Shrimp Lethality Assay and FTIR Analysis of the Extract and Fractions of the Whole Plant of Heliotropium europaeum

Authors: Jahangir Khan Achakzai; Muhammad Anwar Panezai; Basira Akhtar; Shahabuddin Kakar; Ali Akbar; Abdul Manan Kakar; Javed Khan; Nazima Yousaf Khan; Ghulam Mustafa Khan; Muhammad Imran; Marina Panezai; Nisar Ahmed Shahwani; Tehmina Achakzai
Journal: Mediators of Inflammation
 (2020)

---

## Abstract

In this study, anti-inflammatory, anticancer, brine shrimp lethality, and FTIR studies were evaluated. The oxidative burst assay using the chemiluminescence technique, MTT assay, brine shrimp lethality assay, and FTIR analysis were the methods used for the evaluation of anti-inflammatory, anticancer, brine shrimp lethality, and FTIR studies, respectively. The whole-plant butanol fraction ofHeliotropium europaeum (WBFHE) showed anti-inflammatory activity on ROS having IC50 14.7±2.5 while the extract and other fractions of the whole plant of Heliotropium europaeum exhibited no anti-inflammatory activity. None of the extract and fractions of the whole plant of Heliotropium europaeum exhibited anticancer (MCF-7, 3T3, and HeLa cell lines) activities. The whole-plant aqueous fraction of Heliotropium europaeum (WAFHE) and whole-plant butanol fraction of Heliotropium europaeum (WBFHE) showed lethality at high concentration while at low concentration, no toxicity was shown. The whole-plant methanolic extract of Heliotropium europaeum (WMEHE) and whole-plant n-hexane fraction of Heliotropium europaeum (WHFHE) exhibited no toxicity. FTIR interpretation showed the functional groups for the aromatic compounds, phenols, carboxylic acids, esters, alkanes, alkenes, alcohols, alkyl halides, sulfate esters, phosphines, silanes, nitriles, thiols, amines, phosphoric acids, and nitro compounds.


---

## Body

## 1. Introduction

With the beginning of folk medicine, the usage of medicinal plants and their incorporation into allopathic and traditional medicine have a long history [1]. Medicinal plants have effective pharmacological activities due to the presence of secondary metabolites such as alkaloids, saponins, tannins, terpenoids, flavoids, inulin, glycosides, steroids, phlobatannins, terpenoids, phenols, and naphthoquinone. These phytochemicals have less toxicity and side effects [2–5]. The medicinal plant Heliotropium europaeum belongs to the Boraginaceae family, grown in summer, and is a heliotrope [6]. This toxic and medicinal plant is distributed in Middle Eastern countries, for instance, Saudi Arabia, Iraq, Syria, Iran, and Egypt, and Mediterranean countries, for instance, Turkey, Spain, Greece, France, Bosnia, Italy, Albania, Monaco, and Croatia, and is introduced accidently in Australia [7, 8]. Heliotropium europaeum has a poisonous and therapeutic effect, comprises pyrrolizidine which is an important alkaloid, and shows therapeutic properties such as antitumor, insecticidal, hepatotoxic, antibacterial, antifungal, mutagenic, teratogenic, mydriatic, and antispasmodic [9–11]. In this research study, the anti-inflammatory, anticancer (MCF-7, 3T3, and HeLa cell lines), and brine shrimp lethality assay and FTIR studies of the extract and fractions of the whole plant of Heliotropium europaeum are examined.


## 2. Materials and Methods

### 2.1. Plant Materials

The plant material (whole plant) ofHeliotropium europaeum was collected from Dera Bugti, Balochistan, Pakistan, and identified by Prof. Dr. Rasool Bakhsh Tareen and Dr. Shazia Saeed, Department of Botany, University of Balochistan, Quetta, Pakistan, and was deposited in the Herbarium, Department of Botany, University of Balochistan, Quetta, Pakistan, with voucher number QUETTA000016.


### 2.2. Extraction of the Plant Material (Whole Plant) ofHeliotropium europaeum

The whole plant ofHeliotropium europaeum was washed with tap water and then rinsed with distilled water in order to reduce contamination which occurred during transportation and handling and shade dried for one month. This is due to the radiation from sunlight that destroys bioactive compounds present in the whole plant of Heliotropium europaeum. The dried whole plant was grinded in a mechanical grinder, and then, 12 kg powdered plant material was soaked in 20 litres of methanol, kept for 7 days, and shaken daily. After a 7-day period, the methanol-containing whole plant of Heliotropium europaeum was filtered with Whatman filter paper No. 1 and concentrated under reduced pressure at temperature below 55°C in a rotary evaporator. The dried semisolid whole-plant methanolic extract of Heliotropium europaeum (WMEHE) was 288 g. This crude extract 10 g was examined for biological activities such as anti-inflammatory, brine shrimp lethality assay, and anticancer MCF-7 cell line, anticancer 3T3 cell line, and anticancer HeLa cell line activities and FTIR analysis while the remaining extract was fractionated with solvents, for instance, n-hexane, aqueous solution, butanol, ether, dichloromethane, chloroform, and tetrachloromethane [12, 13].


### 2.3. Fractionation of the Crude Extract

The crude extract was fractionated with solvents such as n-hexane, aqueous solution, butanol, ether, dichloromethane, chloroform, and tetrachloromethane to form the whole-plant n-hexane fraction ofHeliotropium europaeum (WHFHE) 3 g, whole-plant aqueous fraction of Heliotropium europaeum (WAFRR) 121.8 g, whole-plant butanol fraction of Heliotropium europaeum (WBFHE) 26 g, whole-plant ether fraction of Heliotropium europaeum (WEFHE) 0.2 g, whole-plant dichloromethane fraction of Heliotropium europaeum (WDFHE) 0.1 g, whole-plant chloroform fraction of Heliotropium europaeum (WCFHE) 0.3 g, and whole-plant tetrachloromethane fraction of Heliotropium europaeum (WTFHE) 0.1 g. The whole-plant n-hexane fraction of Heliotropium europaeum (WHFHE), whole-plant aqueous fraction of Heliotropium europaeum (WAFRR), and whole-plant butanol fraction of Heliotropium europaeum (WBFHE) were examined for anticancer, anti-inflammatory, and brine shrimp lethality assay and FTIT analysis while the whole-plant ether fraction of Heliotropium europaeum (WEFHE), whole-plant dichloromethane fraction of Heliotropium europaeum (WDFHE), whole-plant chloroform fraction of Heliotropium europaeum (WCFHE), and whole-plant tetrachloromethane fraction of Heliotropium europaeum (WTFHE) were examined for FTIR analysis [12, 13].


### 2.4. Anti-Inflammatory Assay

For the anti-inflammatory assay, the oxidative burst assay using the chemiluminescence technique was used.


### 2.5. Oxidative Burst Assay Using the Chemiluminescence Technique

In this technique, 25μl diluted whole blood in HBSS++ containing magnesium chloride and calcium chloride (Sigma, St. Louis, USA) and 25 μl of the extract and fractions of medicinal plants were incubated for 15 min at 37°C in the thermostat chamber of a luminometer (Labsystems, Helsinki, Finland) and then plated in 96-well plates (Costar, NY, USA). Control wells contain HBSS++ and cells while blank wells contain HBSS++. 25 μl luminol (Sigma Chemical Co., St. Louis, MO, USA) and 25 μl serum opsonized zymosan (Sigma Chemical Co., St. Louis, MO, USA) were added into each well. In terms of relative light units, the level of ROS was recorded in a luminometer. In this assay, ibuprofen with IC50 11.2±1.9 is used as a standard drug [14].


### 2.6. MTT Assay (MCF-7 Cell Lines, 3T3 Cell Lines, and HeLa Cell Lines)

The MCF cell line, 3T3 cell line, and HeLa cell line were purchased from the American Type Culture Collection (ATCC). In this assay, Dulbecco’s modified Eagle’s medium containing ten percent fetal bovine serum and two percent antibiotics such as streptomycin with 100μg/ml and penicillin with 100 IU/ml was used for culturing MCF-7 cell lines, 3T3 cell lines, and HeLa cell lines which were then kept in five percent CO2 and incubated at 37°C. MCF-7 cells, 3T3 cells, and HeLa cells were harvested when confluency was developed and 5×104 cells per well were plated in a 96-well flat. After 24 hours, the extract and fractions of medicinal plants with 50 μg/ml were added and then incubated for 48 hours. After incubation, the extract/fractions were removed. To each well, 100 μl with concentration of 0.5 mg/ml MTT was added and kept in an incubator for 4 hours at 37°C. MTT was reduced into formazan crystals which were then dissolved in 100 μl DMSO and was taken at 570 nm absorbance using a microplate reader (SpectraMax Plus, Molecular Devices, CA, USA). In this assay, doxorubicin was used as a standard drug for the MCF-7 cell line and HeLa cell line while cycloheximide was used as a standard drug for the 3T3 cell line. The decrease in viable cells or percent inhibition was calculated with the help of the following formula:
(1)%inhibition=100−meanofO.D.oftestcompound−meanofO.D.ofnegativecontrolmeanofO.D.ofpositivecontrol−meanofO.D.ofnegativecontrol×100.For the calculation of IC50 20 mM stock solution of the extract/fractions, diluted into working solution with 50 μM, and then in order to get less than 50 percent inhibition, working solution is further diluted in serial dilutions. With the help of EZ-Fit5 software, IC50 is calculated [15].


### 2.7. Brine Shrimp Lethality Assay

B-Hatching techniques were used for the evaluation of toxicity.


### 2.8. B-Hatching Techniques

In this B-Hatching technique, scatter 50 mg of brine shrimp eggs in a hatching tray which was already half filled with filtered brine solution. Put it in an incubator at 37°C for 2 days. Take 20 mg of the extract and fractions of medicinal plants, and dissolve it in 2 ml of solvent such as methanol. Transfer 5, 50, and 500μl from this solution to 3 vials, and bring the concentration to 10, 100, and 1000 μg/ml. Overnight, allow the solvent to evaporate. With the help of a Pasteur pipette, put 30 larvae per vial. Add 5 ml seawater. Under illumination, for 24 hours, incubate it at 25-27°C. For positive and negative controls, add a reference cytotoxic drug along with solvent in other vials. Etoposide was the standard drug used in this research study with 7.4625 μg/ml. For the determination of LD50, the Finney computer program was used [16].


### 2.9. FTIR Analysis

The extract and fractions of the plant were dried for the analysis of FTIR. In FTIR analysis, the extract and fractions of the plant with the concentration of 10 mg were encapsulated in the pellet of 100 mg of KBr, for the preparation of the disc of the translucent sample which was then loaded in the FTIR spectroscope (Shimadzu, IRAffinity-1, Japan) [17].


## 2.1. Plant Materials

The plant material (whole plant) ofHeliotropium europaeum was collected from Dera Bugti, Balochistan, Pakistan, and identified by Prof. Dr. Rasool Bakhsh Tareen and Dr. Shazia Saeed, Department of Botany, University of Balochistan, Quetta, Pakistan, and was deposited in the Herbarium, Department of Botany, University of Balochistan, Quetta, Pakistan, with voucher number QUETTA000016.


## 2.2. Extraction of the Plant Material (Whole Plant) ofHeliotropium europaeum

The whole plant ofHeliotropium europaeum was washed with tap water and then rinsed with distilled water in order to reduce contamination which occurred during transportation and handling and shade dried for one month. This is due to the radiation from sunlight that destroys bioactive compounds present in the whole plant of Heliotropium europaeum. The dried whole plant was grinded in a mechanical grinder, and then, 12 kg powdered plant material was soaked in 20 litres of methanol, kept for 7 days, and shaken daily. After a 7-day period, the methanol-containing whole plant of Heliotropium europaeum was filtered with Whatman filter paper No. 1 and concentrated under reduced pressure at temperature below 55°C in a rotary evaporator. The dried semisolid whole-plant methanolic extract of Heliotropium europaeum (WMEHE) was 288 g. This crude extract 10 g was examined for biological activities such as anti-inflammatory, brine shrimp lethality assay, and anticancer MCF-7 cell line, anticancer 3T3 cell line, and anticancer HeLa cell line activities and FTIR analysis while the remaining extract was fractionated with solvents, for instance, n-hexane, aqueous solution, butanol, ether, dichloromethane, chloroform, and tetrachloromethane [12, 13].


## 2.3. Fractionation of the Crude Extract

The crude extract was fractionated with solvents such as n-hexane, aqueous solution, butanol, ether, dichloromethane, chloroform, and tetrachloromethane to form the whole-plant n-hexane fraction ofHeliotropium europaeum (WHFHE) 3 g, whole-plant aqueous fraction of Heliotropium europaeum (WAFRR) 121.8 g, whole-plant butanol fraction of Heliotropium europaeum (WBFHE) 26 g, whole-plant ether fraction of Heliotropium europaeum (WEFHE) 0.2 g, whole-plant dichloromethane fraction of Heliotropium europaeum (WDFHE) 0.1 g, whole-plant chloroform fraction of Heliotropium europaeum (WCFHE) 0.3 g, and whole-plant tetrachloromethane fraction of Heliotropium europaeum (WTFHE) 0.1 g. The whole-plant n-hexane fraction of Heliotropium europaeum (WHFHE), whole-plant aqueous fraction of Heliotropium europaeum (WAFRR), and whole-plant butanol fraction of Heliotropium europaeum (WBFHE) were examined for anticancer, anti-inflammatory, and brine shrimp lethality assay and FTIT analysis while the whole-plant ether fraction of Heliotropium europaeum (WEFHE), whole-plant dichloromethane fraction of Heliotropium europaeum (WDFHE), whole-plant chloroform fraction of Heliotropium europaeum (WCFHE), and whole-plant tetrachloromethane fraction of Heliotropium europaeum (WTFHE) were examined for FTIR analysis [12, 13].


## 2.4. Anti-Inflammatory Assay

For the anti-inflammatory assay, the oxidative burst assay using the chemiluminescence technique was used.


## 2.5. Oxidative Burst Assay Using the Chemiluminescence Technique

In this technique, 25μl diluted whole blood in HBSS++ containing magnesium chloride and calcium chloride (Sigma, St. Louis, USA) and 25 μl of the extract and fractions of medicinal plants were incubated for 15 min at 37°C in the thermostat chamber of a luminometer (Labsystems, Helsinki, Finland) and then plated in 96-well plates (Costar, NY, USA). Control wells contain HBSS++ and cells while blank wells contain HBSS++. 25 μl luminol (Sigma Chemical Co., St. Louis, MO, USA) and 25 μl serum opsonized zymosan (Sigma Chemical Co., St. Louis, MO, USA) were added into each well. In terms of relative light units, the level of ROS was recorded in a luminometer. In this assay, ibuprofen with IC50 11.2±1.9 is used as a standard drug [14].


## 2.6. MTT Assay (MCF-7 Cell Lines, 3T3 Cell Lines, and HeLa Cell Lines)

The MCF cell line, 3T3 cell line, and HeLa cell line were purchased from the American Type Culture Collection (ATCC). In this assay, Dulbecco’s modified Eagle’s medium containing ten percent fetal bovine serum and two percent antibiotics such as streptomycin with 100μg/ml and penicillin with 100 IU/ml was used for culturing MCF-7 cell lines, 3T3 cell lines, and HeLa cell lines which were then kept in five percent CO2 and incubated at 37°C. MCF-7 cells, 3T3 cells, and HeLa cells were harvested when confluency was developed and 5×104 cells per well were plated in a 96-well flat. After 24 hours, the extract and fractions of medicinal plants with 50 μg/ml were added and then incubated for 48 hours. After incubation, the extract/fractions were removed. To each well, 100 μl with concentration of 0.5 mg/ml MTT was added and kept in an incubator for 4 hours at 37°C. MTT was reduced into formazan crystals which were then dissolved in 100 μl DMSO and was taken at 570 nm absorbance using a microplate reader (SpectraMax Plus, Molecular Devices, CA, USA). In this assay, doxorubicin was used as a standard drug for the MCF-7 cell line and HeLa cell line while cycloheximide was used as a standard drug for the 3T3 cell line. The decrease in viable cells or percent inhibition was calculated with the help of the following formula:
(1)%inhibition=100−meanofO.D.oftestcompound−meanofO.D.ofnegativecontrolmeanofO.D.ofpositivecontrol−meanofO.D.ofnegativecontrol×100.For the calculation of IC50 20 mM stock solution of the extract/fractions, diluted into working solution with 50 μM, and then in order to get less than 50 percent inhibition, working solution is further diluted in serial dilutions. With the help of EZ-Fit5 software, IC50 is calculated [15].


## 2.7. Brine Shrimp Lethality Assay

B-Hatching techniques were used for the evaluation of toxicity.


## 2.8. B-Hatching Techniques

In this B-Hatching technique, scatter 50 mg of brine shrimp eggs in a hatching tray which was already half filled with filtered brine solution. Put it in an incubator at 37°C for 2 days. Take 20 mg of the extract and fractions of medicinal plants, and dissolve it in 2 ml of solvent such as methanol. Transfer 5, 50, and 500μl from this solution to 3 vials, and bring the concentration to 10, 100, and 1000 μg/ml. Overnight, allow the solvent to evaporate. With the help of a Pasteur pipette, put 30 larvae per vial. Add 5 ml seawater. Under illumination, for 24 hours, incubate it at 25-27°C. For positive and negative controls, add a reference cytotoxic drug along with solvent in other vials. Etoposide was the standard drug used in this research study with 7.4625 μg/ml. For the determination of LD50, the Finney computer program was used [16].


## 2.9. FTIR Analysis

The extract and fractions of the plant were dried for the analysis of FTIR. In FTIR analysis, the extract and fractions of the plant with the concentration of 10 mg were encapsulated in the pellet of 100 mg of KBr, for the preparation of the disc of the translucent sample which was then loaded in the FTIR spectroscope (Shimadzu, IRAffinity-1, Japan) [17].


## 3. Results

One extract and seven fractions were extracted and fractionated, respectively, from the whole plant ofHeliotropium europaeum. Plant material (gm), yield (gm), and percentage yield of the extract and fractions of the whole plant of Heliotropium europaeum are shown in Table 1.
(1)
Whole-plant methanolic extract ofHeliotropium europaeum (WMEHE)
(2)
Whole-plant n-hexane fraction ofHeliotropium europaeum (WHFHE)
(3)
Whole-plant aqueous fraction ofHeliotropium europaeum (WAFHE)
(4)
Whole-plant butanol fraction ofHeliotropium europaeum (WBFHE)
(5)
Whole-plant ether fraction ofHeliotropium europaeum (WEFHE)
(6)
Whole-plant dichloromethane fraction ofHeliotropium europaeum (WDFHE)
(7)
Whole-plant chloroform fraction ofHeliotropium europaeum (WCFHE)
(8)
Whole-plant tetrachloromethane fraction ofHeliotropium europaeum (WTFHE)Table 1
Plant material (gm), yield (gm), and percentage yield of the extract and fractions of the whole plant ofHeliotropium europaeum.
Plant
Extract and fractions
Plant material
Plant material (gm)
Yield (gm)
Percentage yield%Yield=W1/W2×100W1 = yield (gm)W2 = plant material (gm)
Heliotropium europaeum
WMEHE
Whole plant
12000
288
2.4%
WHFHE
Whole plant
12000
3
0.025%
WAFHE
Whole plant
12000
121.8
1%
WBFHE
Whole plant
12000
26
0.2%
WEFHE
Whole plant
12000
0.2
0.0016%
WDFHE
Whole plant
12000
0.1
0.0008%
WCFHE
Whole plant
12000
0.3
0.0025%
WTFHE
Whole plant
12000
0.1
0.0008%The whole-plant butanol fraction ofHeliotropium europaeum (WBFHE) showed anti-inflammatory activity on ROS having IC50 14.7±2.5 while the extract and other fractions of the whole plant of Heliotropium europaeum exhibited no anti-inflammatory activity. The anti-inflammatory activity of the extract and fractions of the whole plant of Heliotropium europaeum is shown in Table 2.Table 2
Anti-inflammatory activities of the extract and fractions of the whole plant ofHeliotropium europaeum.
S. no.
Extract/fraction/Std. drug
Conc. (μg/ml)
% inhibition/stimulation
IC50±S.D.
1
WMEHE
25
33.2
—
2
WHFHE
250, 50, 10
—
>250
3
WAFHE
250, 50, 10
—
>250
4
WBFHE
250, 50, 10
—
14.7±2.5
5
Ibuprofen
73.2
11.2±1.9μg/mlNone of the extract and fractions of the whole plant ofHeliotropium europaeum exhibited anticancer (MCF-7, 3T3, and HeLa cell lines) activities. Anticancer activities of the extract and fractions of the whole plant of Heliotropium europaeum are shown in Tables 3–5 and Figures 1–4.Table 3
Anticancer assay (MCF-7) of the extract and fractions of the whole plant ofHeliotropium europaeum.
S. no.
Extract/fraction/Std. drug
Conc. (μg/ml)
% inhibition/stimulation
% viability
IC50±S.D.
1
WMEHE
50
10.38
89.62
Inactive
2
WHFHE
50
20.89
79.11
Inactive
3
WAFHE
50
29.19
70.81
Inactive
4
WBFHE
19.96
80.04
Inactive
5
Doxorubicin
50
73.23
26.77
0.80±0.05Table 4
Anticancer assay (3T3) of the extract and fractions of the whole plant ofHeliotropium europaeum.
S. no.
Extract/fraction/Std. drug
Conc. (μg/ml)
% inhibition/stimulation
% viability
IC50±S.D.
1
WMEHE
30
12
88
Inactive
2
WHFHE
30
5
95
Inactive
3
WAFHE
30
0
100
Inactive
4
WBFHE
11
89
Inactive
5
Cycloheximide
30
71
29
0.85±0.07Table 5
Anticancer assay (HeLa cell line) of the extract and fractions of the whole plant ofHeliotropium europaeum.
S. no.
Extract/fraction/Std. drug
Conc. (μg/ml)
% inhibition/stimulation
% viability
IC50±S.D.
1
WMEHE
30
12
88
Inactive
2
WHFHE
30
22
78
Inactive
3
WAFHE
30
3
97
Inactive
4
WBFHE
30
26
74
Inactive
5
Doxorubicin
30
70
30
1.2±0.4Figure 1
Anticancer assay (MCF-7) of the extract and fractions of the whole plant ofHeliotropium europaeum.Figure 2
Anticancer assay (3T3) of the extract and fractions of the whole plant ofHeliotropium europaeum.Figure 3
Anticancer assay (HeLa cell line) of the extract and fractions of the whole plant ofHeliotropium europaeum.Figure 4
Anticancer assay (MCF-7, 3T3, and Hela cell lines) of the extract and fractions of the whole plant ofHeliotropium europaeum.The whole-plant aqueous fraction ofHeliotropium europaeum (WAFHE) and whole-plant butanol fraction of Heliotropium europaeum (WBFHE) showed lethality at high concentration while at low concentration, no toxicity was shown. The whole-plant methanolic extract of Heliotropium europaeum (WMEHE) and whole-plant n-hexane fraction of Heliotropium europaeum (WHFHE) exhibited no toxicity. The results of the brine shrimp lethality assay of the extract and fractions of the whole plant of Heliotropium europaeum are shown in Table 6.Table 6
Brine shrimp lethality bioassay of the extract and fractions of the whole plant ofHeliotropium europaeum.
S. no
Extract and fractions
Dose (μg/ml)
No. of shrimps
No. of survivors
% mortality
LD50 (μg/ml)
Std. drug
LD50 (μg/ml)
1
WMEHE
10
30
30
0%
Etoposide
7.4625
100
30
30
0%
1000
30
30
0%
2
WHFHE
10
30
30
0%
Etoposide
7.4625
100
30
30
0%
1000
30
30
0%
3
WAFHE
10
30
30
0%
Etoposide
7.4625
100
30
30
0%
1000
30
28
6.66%
4
WBFHE
10
30
30
0%
Etoposide
7.4625
100
30
29
3.33%
1000
30
19
36.66%FTIR interpretation of the extract and fractions ofHeliotropium europaeum showed the functional groups for the aromatic compounds, phenols, carboxylic acids, esters, alkanes, alkenes, alcohols, alkyl halides, sulfate esters, phosphines, silanes, nitriles, thiols, amines, phosphoric acids, and nitro compounds. FTIR analysis of the extract and fractions of the whole plant of Heliotropium europaeum is shown in Tables 7–14. FTIR analysis of the extract and fractions of the whole plant of Heliotropium europaeum is shown in Figures 5–12.Table 7
FTIR analysis of the whole-plant methanol extract ofHeliotropium europaeum (WMEHE).
S. no.
Peak
Height
Corr. height
Base (H)
Base (L)
Area
Corr. area
Interpretation
1
509.21
56.905
0.147
511.14
457.13
18.872
0.075
S-S disulfide
2
567.07
56.1
0.103
576.72
563.21
4.82
0.008
C-Br
3
1082.07
60.349
3.539
1132.21
891.11
85.528
0.029
C-O stretch of esters
4
1143.79
58.889
0.029
1155.36
1134.14
8.188
0.472
C-O stretch of ethers
5
1163.08
58.869
0.101
1195.87
1157.29
14.799
0.339
C-H wag (-CH2X)
6
1236.37
59.02
1.042
1280.73
1197.79
31.687
0.021
P-H bending of phosphine
7
1394.53
61.578
0.131
1408.04
1388.75
7.997
0.192
S=O sulfate esters
8
1436.97
61792
1.586
1510.26
1425.4
32.271
0.035
Ar C-C stretch
9
1629.85
60.249
0.435
1635.64
1512.19
44.756
0.005
Ar CH=CHR aromatic alkenes
10
1722.43
59.069
0.196
1724.36
1691.57
12.183
0.569
C=O stretch of esters
11
2162.2
28.864
0.359
2193.06
1977.04
30.999
17.363
Si-H silane
12
2343.51
45.883
14.637
2405.23
2208.49
37.791
90.882
P-H phosphine sharp
13
2933.73
61.877
13.199
3045.6
2407.16
173.618
C-H stretch of alkanes
14
3356.14
59.289
27.719
3786.27
3047.53
214.072
ArO-H H-bonded of phenolsTable 8
FTIR analysis of the whole-plant hexane fraction ofHeliotropium europaeum (WHFHE).
S. no.
Peak
Height
Corr. height
Base (H)
Base (L)
Area
Corr. area
Interpretation
1
709.8
71.892
4.287
794.67
514.99
144.151
8.191
C-H out of plane of aromatics
2
848.68
69.698
0.827
875.68
796.6
40.345
0.504
C-H out of plane of aromatics
3
910.4
69.82
0.326
939.33
877.61
31.969
0.156
N-H wag amines
4
1107.14
72.618
0.505
1136.07
941.26
106.771
1.709
C-N stretch of amines
5
1176.58
72.667
0.338
1327.03
1138
105.055
0.476
C-O stretch of esters
6
1332.81
71.496
0.004
1340.53
1328.95
6.308
0
N-O sym stretch of nitro compound
7
1396.46
72.004
0.174
1415.75
1342.46
40.297
0.101
S=O sulfate esters
8
1448.54
72.57
1.23
1502.55
1417.68
46.721
0.915
Ar C-C stretch
9
1544.98
71.676
1.37
1595.13
1404.48
48.746
0.937
N-O asym stretch of nitro compound
10
1735.93
72.649
15.406
1907.6
1597.06
131.605
17.518
C=O stretch of esters
11
2872.01
68.436
1.523
2899.01
2401.38
200.732
10.737
C-H stretch of alkanes
12
2958.8
69.969
2.214
3138.18
2900.94
118.397
2.981
C-H stretch of alkanes
13
3307.92
67.218
0.013
3311.78
3140.11
82.598
0.112
ArO-H H-bonded of phenols
14
3329.14
67.239
0.01
3336.85
3311.78
12.149
0.001
N-H stretch of aminesTable 9
FTIR analysis of the whole-plant aqueous fraction ofHeliotropium europaeum (WAFHE).
S. no.
Peak
Height
Corr. height
Base (H)
Base (L)
Area
Corr. area
Interpretation
1
503.42
58.347
0.299
507.28
464.84
15.692
0.115
S-S disulfide
2
542
59.12
0.12
553.57
538.14
5.98
0.012
C-Br stretch
3
1085.92
60.482
4.956
1193.94
914.26
104.059
7.441
C-N stretch of amines
4
1236.37
58.967
0.869
1278.81
1195.87
31.726
0.395
C-H wag (-CH2X)
5
1367.53
60.857
0.077
1369.46
1290.38
31.112
0.116
S=O esters
6
1404.18
61.898
0.071
1406.11
1369.40
15.154
0.028
S=O sulfate esters
7
1435.04
62.618
0.128
1438.9
1425.4
5.75
0.012
Ar C-C stretch
8
1543.05
59.517
0.168
1546.91
1510.26
14.182
0.059
N-O asym stretch of nitro compounds
9
1571.99
60.098
0.077
1573.91
1558.48
6.125
0.008
N=O nitroso
10
1631.78
62.413
0.105
1633.71
1575.84
23.792
0.041
NH2 in plane bend of amine
11
1643.35
62.75
0.205
1649.14
1635.64
5.774
0.021
C=N
12
2954.95
56.255
3.937
3026.31
2403.3
169.441
7.483
C-H stretch of alkanes
13
3300.2
58.334
0.054
3304.06
3028.24
100.312
0.428
ArO-H H-bonded of phenols
14
3381.21
58.978
0.035
3385.07
3305.99
30.353
0.027
N-H stretch of aminesTable 10
FTIR analysis of the whole-plant butanol fraction ofHeliotropium europaeum (WBFHE).
S. no.
Peak
Height
Corr. height
Base (H)
Base (L)
Area
Corr. area
Interpretation
1
526.57
60.462
0.202
528.5
455.2
27.591
0.578
S-S disulfide
2
557.43
60.525
0.205
565.14
549.71
6.213
0.02
C-Br
3
787.03
57.485
0.066
792.74
771.53
7.873
0.008
C-Cl
4
1093.64
72.754
7.211
1190.08
896.9
146.453
17.993
C-O stretch of ethers
5
1236.37
72.97
2.73
1296.16
1192.01
56.711
2.085
C-H wag (-CH2X)
6
1375.25
72.012
1.477
1409.96
1298.09
59.987
1.356
CH2 and CH3 of alkanes
7
1440.83
72.733
4.684
1516.05
1417.86
48.245
3.253
S=O sulfate esters
8
1627.92
68.05
0.069
1629.85
1614.42
7.612
0.012
C=N
9
1722.43
71.371
15.773
1840.09
1681.93
54.691
8.927
Monomer C=O of carboxylic acids
10
2058.05
23.659
0.013
2059.98
2011.76
5.615
0.008
N=C in R-N=C=S
11
2945.3
72.963
18.244
3041.74
2407.16
207.79
34.031
C-H stretch of alkanes
12
3367.71
69.56
36.829
3693.68
3043.67
260.725
125.447
Dimer O-H of carboxylic acidsTable 11
FTIR analysis of the WEHE5 whole-plant ether fraction ofHeliotropium europaeum (WEFHE).
S. no.
Peak
Height
Corr. height
Base (H)
Base (L)
Area
Corr. area
Interpretation
1
526.57
67.089
0.244
528.5
414.7
47.877
0.959
S-S disulfide
2
542
67.422
0.24
545.85
530.42
7.488
0.033
C-Br
3
837.11
64.225
6.395
894.97
804.32
37.173
3.73
C-Cl
4
1056.99
85.296
15.749
1168.86
987.55
121.948
29.855
P-H bending of phosphine
5
1228.66
69.684
2.584
1259.52
1170.79
43.885
1.595
C-H wag (-CH2X)
6
1269.16
68.233
0.066
1280.73
1261.451282.66
9.597
0.009
C-O stretch of carboxylic acids
7
1394.53
76.531
2.519
1417.68
1282.66
77.545
3.229
S=O sulfate esters
8
1452.4
80.038
12.112
1575.84
1419.61
70.087
7.949
CH2 and CH3 of alkanes
9
1705.07
62.731
30.359
1832.38
1577.77
71.348
27.206
Dimer C=O of carboxylic acids
10
2036.83
25.82
2.664
2129.41
1982.82
17.65
1.04
N=C in R-N=C=S
11
2183.42
23.243
0.678
2216.21
2131.34
9.575
0.187
Si-H silane
12
2341.58
40.615
13.598
2399.45
2249
25.344
5.458
P-H phosphine sharp
13
2889.37
83.769
1.082
2899.01
2401.38
176.297
0.617
C-H stretch of alkanes
14
2951.09
86.663
9.854
3035.96
2900.94
95.107
11.272
C-H stretch of alkanes
15
3385.07
83.763
45.421
3728.4
3037.89
387.188
210.043
Dimer OH of carboxylic acidsTable 12
FTIR analysis of the whole-plant dichloromethane fraction ofHeliotropium europaeum (WDFHE).
S. no.
Peak
Height
Corr. height
Base (H)
Base (L)
Area
Corr. area
Interpretation
3
509.21
66.099
0.243
518.85
501.49
8.128
0.026
S-S disulfide
4
545.85
65.557
0.072
551.64
540.07
5.353
0.006
C-Br
5
677.01
64.418
0.489
700.16
644.22
24.94
0.179
=CH out of plane of alkene
7
779.24
66.065
0.653
796.6
717.52
36.174
0.265
N-H wag amines
8
831.32
66.341
1.118
896.9
798.53
45.718
0.919
C-H out of plane of aromatics
9
1022.27
70.209
1.238
1045.42
898.83
72.487
1.804
P-H bending of phosphine
10
1128.36
71.605
0.119
1134.14
1047.35
46.475
0.176
C-N stretch of amines
11
1170.79
71.839
0.309
1211.3
1136.07
41.235
0.197
C-O stretch of esters
12
1255.66
71.674
0.417
1307.74
1213.23
51.476
0.325
C-H wag (-CH2X)
13
1377.17
71.741
0.552
1411.89
1309.67
55.549
0.369
CH2 and CH3 of alkanes
14
1448.54
71.855
2.008
1521.84
1421.54
52.27
1.535
Ar C-C stretch of phenols
15
1597.06
86.317
0.075
1598.99
1523.76
36.514
0.088
NH2 in plane bend
16
1612.49
68.717
0.066
1614.42
1598.99
7.742
0.006
C=C stretch of alkenes
17
1724.36
72.514
14.099
1890.24
1616.35
117.083
16.523
C=O stretch of esters
19
2343.51
59.216
9.459
2405.23
2038.76
99.242
5.54
P-H phosphine sharp
20
2573.04
75.36
2.07
2654.05
2407.16
88.15
3.802
S-H sharp of thiols
21
2958.8
69.273
8.105
3061.03
2655.98
177.621
17.639
C-H stretch of alkanes
22
3113.11
63.003
0.016
3115.04
3062.96
22.368
0.011
=C-H stretch of alkenes
23
3307.92
66.138
0.071
331.78
3115.04
89.183
0.536
ArO-H H-bonded of phenols
24
3327.21
66.29
0.02
3329.14
3313.71
7.269
0.001
N-H stretch of amines
28
3402.43
66.579
0.045
3415.93
3394.72
10.093
0.008
N-H stretch of aminesTable 13
FTIR analysis of the whole-plant chloroform fraction ofHeliotropium europaeum (WCFHE).
S. no.
Peak
Height
Corr. height
Base (H)
Base (L)
Area
Corr. area
Interpretation
1
524.64
35.914
3.296
565.14
405.05
27.586
1.549
S-S disulfide
2
611.43
34.252
0.048
617.22
567.07
9.095
0.017
C-Br
3
825.53
39.54
0.956
846.75
727.16
24.686
0.538
N-H wag amines
4
1004.91
53.907
5.508
1037.7
887.26
42.198
3.138
P-H bending of phosphine
5
1101.35
61.8
3.37
1124.5
1039.63
32.25
1.729
C-N stretch of amines
6
1145.72
61.54
1.113
1192.01
1126.43
26.692
0.607
C-O stretch of esters
7
1236.37
61.804
5.301
1305.81
1193.94
42.658
2.845
C-H wag (-CH2X)
8
1363.67
55.831
5.527
1417.68
1307.74
36.288
2.751
C-H rock of alkanes
9
1450.47
51.6
7.497
1535.34
1419.61
29.295
2.774
CH2 and CH3 of alkanes
10
1610.56
43.11
5.439
1656.85
1537.27
26.19
2.202
NH2 in plane bend of amines
11
1722.29
54.706
18.628
2029.11
1658.78
56.075
7.445
C=O stretch of esters
12
2073.48
18.552
0.007
2079.26
2054.19
2.234
0.001
N=C in R-N=C=S
13
2341.58
32.536
13.367
2405.23
2102.41
32.959
5.348
P-H phosphine sharp
14
2563.4
20.744
0.021
2571.11
2515.18
5.636
0.006
S-H sharp of thiols
15
2582.68
20.744
0.025
2611.62
2573.04
3.89
0.003
(O=)PO-H phosphoric acids
16
2951.09
48.718
22.524
3039.81
2613.55
71.029
20.094
C-H stretch of alkanes
17
3387
37.471
20.375
3685.97
3041.74
99.785
42.816
N-H stretch of aminesTable 14
FTIR analysis of the whole-plant tetrachloromethane fraction ofHeliotropium europaeum (WTFHE).
S. no.
Peak
Height
Corr. height
Base (H)
Base (L)
Area
Corr. area
Interpretation
1
516.92
21.117
0.151
526.57
513.07
1.384
0.006
S-S disulfide
2
794.67
16.108
0.01
796.6
781.17
1.175
0.001
C-H out of plane of aromatics
3
800.46
16.113
0.045
958.62
796.6
11.715
0.077
C-H out of plane of aromatics
4
1157.29
18.074
2.67
1271.09
960.55
24.623
2.378
C-O stretch of esters
5
1365.6
14.92
0.202
1369.46
1350.17
1.335
0.011
S=O esters
6
1483.26
15.627
0.503
1490.97
1473.62
1.263
0.026
Ar C-C stretch of aromatics
7
1517.98
16.033
1.015
1548.84
1510.26
2.791
0.148
N-O asym stretch of nitro compounds
8
1616.35
12.206
0.149
1618.28
1598.99
1.046
0.016
C=N
9
1716.65
15.575
0.436
1722.43
1703.14
1.383
0.024
C=O of carboxylic acids
10
1730.15
15.697
0.748
1799.59
1724.36
4.615
0.069
C=O stretch of esters
11
2239.36
8.703
0.111
2249
2214.28
1.358
0.008
Si-H silane
12
2929.87
13.999
7.508
3053.32
2725.42
13.854
4.244
C-H stretch of alkanes
13
3078.39
6.297
0.044
3136.25
3072.6
1.761
0.01
Ar-H stretch of aromatics
14
3188.33
6.016
0.004
3190.26
3153.61
0.981
0
Dimer O-H of carboxylic acidsFigure 5
FTIR spectrum analysis of the whole-plant methanol extract ofHeliotropium europaeum (WMEHE).Figure 6
FTIR spectrum analysis of the whole-plant hexane fraction ofHeliotropium europaeum (WHFHE).Figure 7
FTIR spectrum analysis of the whole-plant aqueous fraction ofHeliotropium europaeum (WAFHE).Figure 8
FTIR spectrum analysis of the whole-plant butanol fraction ofHeliotropium europaeum (WBFHE).Figure 9
FTIR spectrum analysis of the whole-plant ether fraction ofHeliotropium europaeum (WEFHE).Figure 10
FTIR spectrum analysis of the whole-plant dichloromethane fraction ofHeliotropium europaeum (WDFHE).Figure 11
FTIR spectrum analysis of the whole-plant chloroform fraction ofHeliotropium europaeum (WCFHE).Figure 12
FTIR spectrum analysis of the whole-plant tetrachloromethane fraction ofHeliotropium europaeum (WTFHE).


## 4. Conclusion

In this research study, the whole-plant butanol fraction ofHeliotropium europaeum (WBFHE) showed anti-inflammatory activity on ROS having IC50 14.7±2.5 while the extract and other fractions of the whole plant of Heliotropium europaeum exhibited no anti-inflammatory activity. None of the extract and fractions of the whole plant of Heliotropium europaeum exhibited anticancer (MCF-7, 3T3, and HeLa cell lines) activities. The whole-plant aqueous fraction of Heliotropium europaeum (WAFHE) and whole-plant butanol fraction of Heliotropium europaeum (WBFHE) showed lethality at high concentration while at low concentration, no toxicity was shown. The whole-plant methanolic extract of Heliotropium europaeum (WMEHE) and whole-plant n-hexane fraction of Heliotropium europaeum (WHFHE) exhibited no toxicity. FTIR interpretation showed the functional groups for the aromatic compounds, phenols, carboxylic acids, esters, alkanes, alkenes, alcohols, alkyl halides, sulfate esters, phosphines, silanes, nitriles, thiols, amines, phosphoric acids, and nitro compounds.



---

*Source: 5056897-2020-02-01.xml*