Effect of Senna plant on the mitochondrial activity of Hymenolepis diminuta

Abstract

The peculiarity of energy metabolism in helminths is the ability to undergo transition from aerobic to anaerobic under low oxygen tension. during its adult stage. Fumarate reductase and succinate dehydrogenase of mitochondria are the two enzymes responsible during this transition and adaptation to this hypoxic environment. Earlier we had reported that three species of Senna plant, S. alata, S. alexandrina and S. occidentalis altered the morphology, ionic concentration and neurotransmission of the cestode parasite Hymenolepis diminuta. The present study aimed at exploring the mechanism of leaf extracts of the three plant species of Senna on the mitochondrial activity of the parasite that chiefly involve the NADH-fumarate reductase system which is the terminal step in phosphoenolpyruvate carboxykinase succinate pathway. The structure of mitochondria was observed through electron microsopy and its density was detected through confocal microscopy, spectroflourimetry and spectrophotometry, while enzyme activities were assayed through native gel and spectrophotometric assays. Praziquantel was tested on the parasites as a reference drug to compare its effects with that of the plant extracts. The mitochondria architecture was altered, and enzymes activity decraeased by 60% in all three plant species of Senna treated parasites which suggested that these three Senna species posses potent chemotherapeutic properties.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12639-021-01415-9.

Introduction

Hymenolepiasis is a neglected tropical disease caused by the zoonotic tapeworms Hymenolepis diminuta (rat tapeworm) and H. nana (dwarf tapeworm) which manifest clinical symptoms like diarrhea, abdominal pain, anorexia, and gastrointestinal problems and even causing life threats for immunosuppressed individuals, and thus make their transmission a matter of concern (Cheng et al. 2016; Karuna and Khadanga 2013). Therefore finding an important limiting factor in the parasite as chemo therapeutic target is necessary.

The peculiarity of energy metabolism in adult stage helminths is the ability to undergo transition from aerobic to anaerobic under low oxygen tension, characterized by the reduction of fumarate to succinate depicts the presence of unique parasite biochemical pathway in the mitochondria thus adapting to microaerophillic condition (Van Hellemond et al. 1995; Kita and Takamiya 2002). Fumarate play an essential role in the regeneration of NAD⁺ in adult Ascaris suum (Kita et al. 2007). Membrane-associated fumarate reduction is accomplished with rhodoquinone (RQ) as an electron carrier, proton pumping, and chemiosmotic ATP synthesis (Tielens et al. 2002). Electrons from NADH are transferred to rhodoquinone (RQ) via complex I by NADH rhodoquinone oxidoreductase and from RQ the electrons are transferred to fumarate in complex II, which is then reduced to succinate by the enzyme fumarate reductase (Saruta et al. 1995; Omura et al. 2001). Reduction of fumarate to succinate is unique in the mitochondria respiratory system of helminth parasites (Kita and Takamiya, 2002). Succinate dehydrogenase enzyme is homologous to fumarate reductase that catalyze the reverse reaction during anaerobic respiration in bacteria (Hägerhäll 1997). The enzyme plays an essential role in aerobic respiratory chain in mitochondria whereas fumarate reductase catalyzes the final step in anaerobic respiration with fumarate as a terminal electron acceptor and thus enzyme complexes in this cell organelle is defined as examples of the high evolutionary adaptation of organisms to specialized environments (Kröger 1978; Hägerhäll 1997). Fumarate is a major pathway adapted by parasites for generating energy during hypoxic environment, and predicted as one of the most promising targets for chemotherapy (Matsumoto et al. 2008; Sakai et al. 2012).

Few drugs such as albendazole, oxamniquine, praziquantel, ivermectin, diethylcarbamazine and mebendazole were found to combat common helminth infections across the world (Hotez 2009; Cotreau et al. 2003), but some of these drugs have shown resistance and thus poses a major concern to chemotherapy (Seto et al. 2011). Extensive research activity on different plant species and their therapeutic principles with lesser side effects compared to allopathic medicines were re-evaluated (Kala 2005). Apart from their utility in treatment of diseases, use of medicinal plants has been a practice for trading in herbal markets as well (Ahmad et al. 2018; Segun et al. 2018).

Leaf extracts of Coccinia grandis, Carex baccans and phytochemicals of Securinega were reported to have caused changes in the architecture of the mitochondria of cestode parasite Raillietina echinobothrida (Challam et al. 2012; Dasgupta et al. 2013; Giri and Roy 2014). The three species of Senna plants (synonym of Cassia) viz. S. alata Linn., S. alexandrina Mill. and S. occidentalis (L) Link. were earlier reported by our team which altered the ionic homeostasis of the parasite and significantly affected the activity and expression of acetylcholinesterase and serotonin (Kundu et al. 2012, 2015 and 2016, Roy et al. 2016; Ukil et al. 2018a). It is thus intriguing to continue to understand the mechanism of action of these plant extracts on the mitochondrial activity of the parasite if altered during the process of paralysis.

Materials and methods

Collection and preparation of plant extracts

Crude ethanolic extract from three species of Senna plant was prepared in the same way as described earlier (Kundu and Lyndem 2012).

Collection and treatment of parasites

H. diminuta was obtained from our laboratory raised male Albino rats in the method described earlier by Kundu et al. (2012). About 5 live worms were incubated in 40 mg/mL concentration of each of the three plant leaf extracts and in 0.005 mg/mL praziquantel prepared in phosphate buffer saline (PBS) and 1% dimethylsulphoxide (DMSO) with one set of worms as control prepared by only PBS and DMSO. After the worms were paralysed, they were collected for different experimental analysis. The concentrations prepared were derived after standardiization from our earlier studies after testing with different plant concentrations. All experimental protocols with rats were approved by the Institutional Animal Ethics Committee (IAEC), Visva-Bharati University letter No IAEC/VB/2017/06.

Drugs and chemicals

All salts and reagents were obtained from Merck, USA and Hi-media Laboratories, India. The primary fixative gluteraldehyde was obtained from Merck, USA. Sodium cacodylate, decyl ubiquinone (dUQ) and acetyl coenzyme A (acetyl CoA) were supplied by Sigma Aldrich, while Mitotracker chloromethyl tetramethylrosamine (CMTMRos) was obtained from Invitrogen, Thermo Fischer Scientific, India. Sucrose monolaurate was obtained from Santa Cruz, California, USA. Bovine serum albumin (BSA) and HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were obtained from Hi-media Laboratories, India. Chemicals for native gel electrophoresis, Coomasie brilliant blue R250, dithiobis nitrobenzoic acid (DTNB) or Ellman’s reagent and ethylene glycol-bis [(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)] (EGTA) were obtained from Sisco Research Laboratories. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT), and phenazine methosulphate (PMS) were procured from Hi-media Laboratories, India. Ethanol was supplied by Bengal Chemicals (Kolkata, India). The reference drug praziquantel was purchased from Chandrabhagat Pharma Pvt Ltd., Mumbai, India.

Preparation of parasites for transmission electron microscopy (TEM)

We have reported earlier that the three species of Senna altered the morphological structure of the mitochondria (Ukil et al. 2018b). In the present study we compared the plant treated worms with the positive control (praziquantel). After paralysis, all the experimental worms were first washed in PBS for 5–10 min and later fixed in primary fixative (3% gluteraldehyde) for 10 min. The worms were removed and kept in fresh gluteraldehyde for the next 24 h after which they were transferred to 0.2 M cacodylate buffer and kept till further processing for TEM studies following the method of Dykstra and Reuss (1992). In brief, the worms were cut into small sections (1–1.5 mm each) and dehydrated through graded acetone. The sections were embedded and polymerized with a resin embedding medium and blocks were prepared. Ultrathin sections were obtained through cutting in an ultramicrotome, stained with uranyl acetate and examined under transmission electron microscope (JEOL JEM 100-CX-II).

Isolation of mitochondria

Mitochondria was isolated following Wieckowski et al. (2009) with minor modifications. In brief, three isolation buffers (IB), IB-1, IB-2 and IB-3 were prepared. IB-3 was prepared with 225 mM mannitol, 75 mM sucrose and 30 mM TRIS base in distilled water (pH-7.4). IB-1 was prepared by adding 0.5% BSA and 0.5 M EGTA in IB-3 (pH-7.4) while IB-2 was prepared by adding 0.5% BSA to IB-3. Mitochondrial resuspension buffer (MRB) was prepared through mixture of 225 mM mannitol, 5 mM HEPES buffer and 0.5 mM EGTA.

About 0.5 g parasite tissue was weighed and homogenized at 1,500 r.p.m in 1 mL of IB-1 at 4 °C and then subjected to differential centrifugation where the homogenized tissue was centrifuged (Remi CM-12 plus cooling centrifuge) at 750 g for 5 min at 4 °C two times after every supernatant collection, and further centrifuged at 9000 g for 10 min at 4 °C after which mitochondria was separated as pellet and cytosol was obtained in the supernatant. The mitochondrial pellet was suspended in IB-2 and centrifuged at 10,000 g for 10 min at 4 °C and the pellet obtained from here was further suspended in IB-3 and centrifuged at 10,000 g for 10 min at 4 °C. The final supernatant was discarded and mitochondrial pellet was collected and used for further analysis.

Detection of mitochondria

Mitochondria was detected through staining method using mitotracker (CMTMRos) according to the manufacturer of Invitrogen product data sheet for detecting mitochondria. In brief, the obtained mitochondrial pellet (25 mg) was suspended in 100 µL MRB. About, 50 µL of the suspension as well as the cytosolic supernatant was pipetted out and 1 µL of dye solution was added to each in such a way that the concentration of the dye becomes 100 nM. This mixture was then incubated for 30 min at room temperature. About 1 µL of this incubated mixture was mounted on glass slides and observed under confocal laser scanning microscope (Leica TCS SP8) at 40 X magnification.

Further detection of mitochondria was performed following the protocol of Scorrano et al. (1999) with some modifications. Another set of final mitochondrial pellet (25 mg) was dissolved in 100 µL MRB after which, 50 µL of this suspension was taken and 1 µL of mitotracker dye was added to it and incubated for 30 min. About 5 µL of this final mixture was added to 3 mL PBS and observed under spectrofluorometer (Perkin Elmer LS-55) and spectrophotometer (Shimadzu UV-3101 PC) at a wavelength range of 554–575 nm. One set of negative control containing only 5 µL of the dye and 3 mL PBS was also observed as positive control.

Functional biogenesis of isolated mitochondria

To confirm the functional biogenesis of mitochondria, different enzyme activities were studied through biochemical enzyme assay and staining method.

Citrate Synthase Activity

The activity of citrate synthase was estimated following the method of Clark et al. (1997). In brief, a reaction buffer was prepared that contained 100 µmol/L acetyl CoA and 10 µmolL⁻¹ DTNB. This reaction buffer was added to 100 mmol/L TRIS buffer (pH-8) and 30 µL of the final mitochondrial suspension to produce a reaction mixture. To initiate the reaction, 10 mmol/L potassium oxalate was added to 20 µL of this reaction mixture and the absorbance was observed at 412 nm in spectrophotometer (Beckman Coulter 730 DU UV–Visible). The specific activity of the enzyme in mmoles of coenzyme A released per minute per milligram protein was calculated using an extinction coefficient of DTNB at 412 nm (13,600 M⁻¹ cm ⁻¹) as follows,

$$Activity=\frac{\mathit{Absorbance}/min}{ExtinctionCoefficient\times Pathlengthincm}$$ 1

Native polyacrylamide gel electrophoresis (Native PAGE)

Native PAGE was performed using a discontinuous gel system method of Davis (1964). Gel was casted with 4% stacking gel and 8% resolving gel in a vertical gel apparatus. About 70 µg of the mitochondrial suspension of control and treated samples were loaded in the gel. To start electrophoresis using Bio-Rad mini protein tetrapack, the gel was connected to a a power supply of 40 mV at which proteins were stacked and then the voltage was increased to 70 mV where the protein bands got separated.

Coomasie staining

A profiling for detection of the total protein content in the mitochondrial fraction was done through coomasie brilliant blue staining following the protocol of Malik and Berrie (1972). In brief, the gel was prepared as that of native PAGE, the final mitochondrial suspension was loaded in the gel and incubated with coomasie stain for overnight and then washed in destainer (40% methyl alcohol and 10% acetic acid) for about 3–4 h and observed in Bio-Rad Gel Doc EZ imager using coomasie filter.

Mitochondrial enzyme activities

• (i)
  Detection of the mitochondrial enzymes were performed through staining process using native PAGE. The native gel for each enzyme was incubated in media containg the different substrates following Singh et al (2006) with minor modifications. In brief, a reaction buffer composed of 30 mM potassium phosphate buffer at pH-7.4 and 1 mM MgCl₂ was prepared. The substrate was added to this reaction buffer and the native gel was incubated for 1 h at room temperature and observed in a Gel Doc imager using EtBr ultraviolet (UV) filter. The concentration of the components of reaction buffer and the substrates were prepared following the method of Matsumoto et al. (2008) as follows:

  1. NADH-fumarate reductase activity was determined using 100 µM NADH and 5 mM fumarate substrate solution.
  2. Succinate dehydrogenase activity was determined by incubating the native gel in 10 mM succinate solution in presence of 60 µg/mL of MTT and 120 µg/mL PMS.
  3. NADH-quinone reductase activity was detected by using 100 µM NADH and 60 µM dUQ substrate solutions.
  4. Succinate-quinone reductase activity was stained using 0.1% weight/volume of sucrose monolaurate solution in the incubation medium.
  5. NADH oxidase activity staining was performed using 100 µM NADH substrate solution as the incubation medium.

• (ii)
  Estimation of the activity of the mitochondrial enzymes was performed biochemically following Matsumoto et al. (2008). In brief, a reaction medium was prepared that composed of 100 µg/mL glucose oxidase, 2 µg/mL catalase, and 10 mM β-D-glucose. This medium was added to the reaction buffer (same as prepared for native gel staining procedure) and kept for 3 min to achieve anaerobiosis as the remaining oxygen is removed by the reactions catalyzed by glucose oxidase and catalase. To this reaction medium, respective enzyme substrate was added to produce a final reaction mixture volume of 1 mL. The final mitochondrial protein concentration in this 1 mL reaction mixture was 80 µg/mL. This concentration was derived from 30 µL of the pellet suspension. The substrate for each enzyme estimation was added in similar concentrations as that of native gel staining method. The activity of each enzyme was obtained as the rate of change of enzyme activity at a regular time interval.

  1. NADH fumarate reductase activity was determined by monitoring the oxidation of NADH at 340 nm in UV–visible spectrophotometer. The reaction was initiated by adding NADH solution and the specific activity of the enzyme was calculated as follows:

     $$\begin{array}{c}Changeinabsorbance\left(\mathrm{\Delta }O{D}_{340nm}/min\right)=\frac{Initialreading-Finalreading}{\text{Re}actiontime\left(min\right)}\\ Enzymeactivity\left(\mu mol/min\right)=\frac{\left(\mathrm{\Delta }ODoftestsample-\mathrm{\Delta }ODofblank\right)\times totalreactionvolume\left(\mathit{mL}\right)}{MolarextinctioncoefficientofNADHat340nm\times enzymesourcevolume\left(\mathit{mL}\right)}\\ \end{array}$$ 2

     (the extinction coefficient for NADH at 340 nm for test in 1 mL cuvette = 6.2 mM⁻¹ cm⁻¹. 1 unit = µmol⁻¹ min ⁻¹ specific activity = unit activity mg protein⁻¹).
  2. Succinate dehydrogenase enzyme activity was determined by monitoring the absorbance change MTT in presence of PMS at 570 nm. The reaction was initiated by adding succinate solution and the specific activity of the enzyme was calculated as follows:

     $$\begin{array}{c}changeinabsordance\left(\mathrm{\Delta }O{D}_{570nm}/min\right)=\frac{Initialreading-Finalreading}{\text{Re}actiontime\left(min\right)}\\ Enzymeactivity\left(\mu mol/min\right)=\frac{\left(\mathrm{\Delta }ODoftestsample-\mathrm{\Delta }ODofblank\right)\times totalreactionvolume\left(\mathit{mL}\right)}{Molarextinctioncoefficientoftetrazoliumat570nm\times enzymesourcevolume\left(\mathit{mL}\right)}\\ \end{array}$$ 3

     (the extinction coefficient of tetrazolium at 570 nm for test in 1 mL cuvette = 13 mM⁻¹ cm⁻¹. 1 unit = µmol⁻⁻¹ min ⁻¹ specific activity = unit activity mg protein⁻¹).
  3. The absorbance change of NADH was observed at 340 nm in the presence dUQ. The reaction was initiated by adding NADH and the specific activity of the enzyme was calculated as follows:

     $$\begin{array}{c}changeinabsordance\left(\mathrm{\Delta }O{D}_{340nm}/min\right)=\frac{Initialreading-Finalreading}{\text{Reaction}time\left(min\right)}\\ Enzymeactivity\left(\mu mol/min\right)=\frac{\left(\mathrm{\Delta }ODoftestsample-\mathrm{\Delta }ODofblank\right)\times totalreactionvolume\left(\mathit{mL}\right)}{MolarextinctioncoefficientofNDAHat340nm\times enzymesourcevolume\left(\mathit{mL}\right)}\\ \end{array}$$ 4

     (the extinction coefficient for NADH at 340 nm for test in 1 mL cuvette = 6.2 mM⁻¹ cm⁻¹. 1 unit = µmol⁻⁻¹ min ⁻¹ specific activity = unit activity mg protein⁻¹).
  4. Succinate quinone reductase activity was determined by measuring the amount of dUQ at 278 nm in presence of sucrose monolaurate. The specific activity of the enzyme was calculated as follows:

     $$\begin{array}{c}changeinabsordance\left(\mathrm{\Delta }O{D}_{278nm}/min\right)=\frac{Initialreading-Finalreading}{\text{Reaction}time\left(min\right)}\\ Enzymeactivity\left(\mu mol/min\right)=\frac{\left(\mathrm{\Delta }ODoftestsample-\mathrm{\Delta }ODofblank\right)\times totalreactionvolume\left(\mathit{mL}\right)}{MolarextinctioncoefficientofdUQat278nm\times enzymesourcevolume\left(\mathit{mL}\right)}\\ \end{array}$$ 5

     (the extinction coefficient for dUQ or test in 1 mL cuvette = 12.7 mM⁻¹ cm⁻¹. 1 unit = µmol⁻⁻¹ min ⁻¹ specific activity = unit activity mg protein⁻¹).
  5. NADH oxidase activity was observed at 340 nm.The reaction was initiated by adding NADH solution.The specific activity of the enzyme was calculated as follows:

     $$\begin{array}{c}changeinabsordance\left(\mathrm{\Delta }O{D}_{340nm}/min\right)=\frac{Initialreading-Finalreading}{\text{Reaction}time\left(min\right)}\\ Enzymeactivity\left(\mu mol/min\right)=\frac{\left(\mathrm{\Delta }ODoftestsample-\mathrm{\Delta }ODofblank\right)\times totalreactionvolume\left(\mathit{mL}\right)}{MolarextinctioncoefficientofNADHat340nm\times enzymesourcevolume\left(\mathit{mL}\right)}\\ \end{array}$$ 6

     (the extinction coefficient for NADH at 340 nm for test in 1 mL cuvette = 6.2 mM⁻¹ cm⁻¹. 1 unit = µmol⁻⁻¹ min ⁻¹ specific activity = unit activity mg protein⁻¹).

Protein estimation

The total protein content of the isolated mitochondrial fraction was determined by Lowry et al. (1951) with Folin Ciocalteu reagent. In brief, 5 µL of the suspended mitochondrial fraction of each of the control and treated parasites was added to 120 µL of distilled water. To this mixture, 1.25 mL of this Lowry’s reagent was added and incubated at 37 °C for 10 min. To the latter, 125 µL of Folin Ciocalteu reagent was added and this mixture was further incubated at 37 °C for 30 min and the absobance of total protein content in the mitochondrial sample was determined at 660 nm.

Statistical analysis

All experiments were repeated for five times (n = 5) and data expressed as the mean ± standard error mean (SEM). The data obtained was analysed by one-way analysis of variance (ANOVA) and was statistically expressed as significant at p value ≤ 0.05 and marked as ‘^*’, p value ≤ 0.01 and marked as ‘^**’, p value ≤ 0.001 and marked as ‘^***’ and p value ≤ 0.0001 and marked as ‘^****’.

Results

Transmission electron microscopy

TEM micrographs showed clustered mitochondria, each with distinct cristae in the control (Fig. 1a) the structure of which was more noticeable at a higher magnification (Fig. 1b). In treated worms, the number of cristae were reduced and the matrix was lucid (Fig. 1c). Such alterations were clearly noticed at higher magnification (Fig. 1d).

Fig. 1.

Transmission electron micrographs showing ultrastructure of mitochondria of H. diminuta. a Control showing cluster of intact mitochondria; c Praziquantel treatment showing mitochondria with indistinct cristae; e S. alata treatment showing deformed mitochondria and cristae; g S. alexandrina treatment showing damage in the mitochondrial membrane and cristae; i S. occidentalis treatment showing distortion of mitochondrial membrane architecture. All magnifications at 8000X with 0.2 µm scale bar. b Control showing distinct mitochondrial membrane, cristae and matrix: d Praziquantel showing absolute distortion in the mitochondrial membrane and highly dense matrix with indistinct cristae even at a higher magnification; f S. alata showing a distinct distortion in mitochondrial membrane and damaged cristae with clumped matrix; h S. alexandrina showing distorted mitochondrial membrane with an indisctinct architecture of cristae and lucid matrix; j S. occidentalis showing distorted mitochondrial membrane and cristae with a lucid matrix in the mitochondria. All at a higher magnification of 20000X with 100 nm scale bar

Confocal microscopy studies showed fluorescence dots in the mitochondrial fraction of both control and treated parasites (Fig. 2a–e). However no fluorescence was observed in the cytosolic fraction (Supplementary Fig. 1).

Fig. 2.

Confocal micrographs of isolated mitochondria pellet of H. diminuta. a Control showing deep stained fluorescence dots; b Praziquantel showing fluorescence at par with the control; c S. alata showing a slight decrease in fluorescenc; d S. alexandrina less intense fluorescence dots; e S. occidentalis showing decreased intensity of fluorescence. All magnifications at 40X

Observation of mitochondria through Spectrofluorimetric and UV Spectrophotometric studies

Spectrofluorimetry study showed the excitation of mitotracker dye at 560 nm with a maximum emission band at 580 nm and a maximum excitation at 570 nm (Fig. 3a). There was no shift in the excitation maxima after addition of mitochondrial sample. However, fluorescence quenching of mitotracker was observed and fluorescence intensity was reduced from 140 arbitrary units (au) to 80 au (Fig. 3b).

Fig. 3.

Spectrofluorimetric and UV Spectrophotometric studies of mitochondrial fraction of H. diminuta. a Fluorescence exhibited by mitotracker dye at 570 nm excitation maxima in spectrofluorometer; b Fluorescence quenching after addition of mitochondrial fractions from 140 to 80 AU; c Maximum absorbance exhibited by mitotracker dye at 550 nm in UV spectrophotometer; d Absorbance observed at 550 nm with mitochondrial fraction showing absorbance peak of control and S. occidentalis at the same intensity while that of S. alata, S. alexandrina and praziquantel were observed at a different intensity

In case of UV spectrophotometric study, mitotracker dye showed a maximum absorbance at 550 nm (Fig. 3c). The maximum absorbance persisted at a similar wavelength when mitochondrial sample was added to the dye but in the treated worms the absorbance was decreased from that of the control (Fig. 3d).

Observation of enzyme activity in native gel staining

Various mitochondrial protein bands were seen separated within the coomasie gel that indicated the presence of various proteins in the isolated mitochondrial fraction (Fig. 4a). Though expression of NADH-fumarate reductase activity was observed in all the treated parasites, however S. occidentalis treated worms showed less expression compared to that of the control (Fig. 4b). Similarly, succinate dehydrogenase enzyme was clearly noticeable in the mitochondrial fraction of control parasites and low expression was seen in S. occidentalis among the treated parasites (Fig. 4c). Expression of NADH-quinone reductase activity was low in all plant treated groups compared to that of the control (Fig. 4d) while the expression of succinate-quinone reductase was observed to be low in S. alexnadrina compared to that of the control (Fig. 4e). Expression of NADH oxidase activity was decreased in all treated groups from that of the control (Fig. 4f).

Fig. 4.

The enzymes from isolated mitochondrial fraction of H. diminuta in native PAGE. a Coomasie brilliant blue stain, arrows indicating the positions of band formation by different mitochondrial proteins; b NADH-fumarate reductase staining showing low expression in S. occidentalis treated parasites; c Succinate dehydrogenase showing low expression in S. occidentalis treated parasites; d NADH-quinone reductase demonstrate low expression in S alata, S.alexandrina and S.occidentalis treated parasites; e Succinate-quinone reductase expression is low S. alexandrina and S. occidentalis treated parasites; f NADH oxidase enzyme showing low expression in all plant treated parasites

Variations in the activity of mitochondrial enzymes

A significant decrease in citrate synthase, NADH-fumarate reductase, succinate dehydrogenase, NADH-quinone reductase, succinate-quinone reductase and NADH oxidase activities was observed in all the treated worms compared to that of the control (Table 1).

Table 1.

Specific activity of mitochondrial proteins of H. diminuta after treatment with Senna leaf extracts

Enzymes	Specific activity of mitochondrial enzymes in µmol min−1 mg protein−1
	Control	Praziquantel	S. alata	S. alexandrina	S. occidentalis
Citrate synthase	13.6 ± 0.61	7.6*** ± 1.19	4**** ± 0.51	5.2**** ± 0.75	3.1**** ± 0.47
NADH-fumarate reductase	53.1 ± 8.1	26.3** ± 3.76	10**** ± 0.25	19.2**** ± 0.84	20*** ± 0.98
Succinate dehydrogenase	22.3 ± 2.95	8.7**** ± 0.56	5.2**** ± 0.43	13.2** ± 0.75	7.4**** ± 0.47
NADH-quinone reductase	56.7 ± 2.05	31.6**** ± 3.17	13.2**** ± 2.3	22**** ± 8.4	16.4**** ± 2.41
Succinate-quinone reductase	77.6 ± 8.01	37**** ± 2.96	6.7**** ± 0.61	14.0**** ± 0.61	20.7**** ± 0.70
NADH oxidase	28.4 ± 2.13	11.2**** ± 0.60	9.3**** ± 2.07	10.1**** ± 0.236	9.9**** ± 1.89

Decrease in specific activities of mitochondrial enzymes in treated H. diminuta shown as a mean value ± SEM (n = 5). All values indicate a decrease in enzyme activities in the mitochondria of treated parasites from that of the control which are: ^**significant at a p < 0.01; ^*** significant at p < 0.001; ^**** significant at p < 0.0001; ns represent non-significant change

Change in the activity of citrate synthase

Citrate synthase was observed as 13.6 ± 0.61 µmol min⁻¹ mg protein⁻¹ in the mitochondria of control parasites but decreased significantly in all the treated parasites. The enzyme decreased by 77.2% in S. occidentalis followed by S. alata (70.58%), S. alexandrina (61.76%) and praziquantel (44.11%).

Change in the activity of NADH-fumarate reductase

NADH-fumarate reductase activity decreased significantly in the mitochondria of all the treated groups from that of the control parasites. It was 53.1 ± 8.1 µmol min-¹ mg protein⁻¹ in control, but decreased by 81.16% in S. alata treated parasites followed by S. alexandrina (63.84%), S. occidentalis (62.33%) and praziquantel (50.47%).

Change in the activity of Succinate dehydrogenase

In the conrol parasite, the activity of succinate dehydrogenase in mitochondria was found to be 22.3 ± 2.95 µmol min⁻¹ mg protein⁻¹ in control. The enzyme activity showed 76.68% reduction in S. alata treated parasites followed by S. occidentalis (66.81%), praziquantel (60.98%) and S. alexandrina (40.80%).

Change in the activity of NADH-quinone reductase

NADH-quinone reductase activity in control parasite was 56.7 ± 2.05 µmol min⁻¹ mg protein⁻¹, while S. alata showed 76.71% reduction from control followed by S. occidentalis (71.07%), S. alexandrina (61.19%) and praziquantel (44.26%).

Change in the activity of Succinate-quinone reductase

Succinate-quinoine reductase was observed to be 77.6 ± 8.01 µmol min⁻¹ mg protein⁻¹ in the control parasite, but decreased significantly by 91.36% in S. alata, followed by S. alexandrina (81.95%), S. occidentalis (73.32%). and praziquantel (52.31%).

Change in the activity of NADH oxidase

NADH oxidase activity in mitochondria was observed as 28.4 ± 2.13 µmol min⁻¹ mg protein⁻¹ in control parasite. All the three plant species showed almost equally significant decrease in enzyme activity from that of the control with S. alata showed 67.25% decrease in enzyme activity followed by S. occidentalis (65.14%), S. alexandrina (64.43%) and praziquantel (60.56%).

Discussion

Mitochondria play crucial role in many cellular processes, including ATP production through oxidative phosphorylation and exhibit diverse morphology related with its functioned state (Cogliati et al. 2016). Alteration in the architecture was observed in the mitochondria of all treated parasites in the present study. The loss of membrane uniformity observed in our study might be an indication of the loss of membrane potential. The mitochondrial membranes allow the exchange of ions and harbours the primary enzyme such as NADH-fumarate reductase along with quninone reductase, succinate dehydrogenase and NADH oxidase for electron transport during energy metabolism with cristae being related to ATP synthesis. These enzymes are usually associated with the mitochondrial membrane and hence the damage in mitochondrial membrane could inhibit electron transport and reduce energy production in the parasite. Further in our present study inhibition of these enzyme activities were confirmed through biochemical assay.

The expression of fluorescence dots indicates binding of the mitochondria with the dye even after the loss of membrane potential and therefore confirmed the presence of mitochondria in the isolated fraction. This was also reported by Kholmukhamedov et al. (2013). The confirmation of the presence of mitochondria thus assured our study on enzyme activities in mitochondrial fraction.

Spectrofluorimetric analysis was performed to confirm the loss of membrane potential of the parasite mitochondria. The fluorescence peak dropped from 140 to 80AU indicated that quenching of fluorescence had occurred. This could be due to the lost of protein activity as the intrinsic fluorophore NADH fluorescent upon binding with protein and increases its emission four fold. This was also observed by Breton (1996) when NADH bound to 17β hydroxysteroid dehydrogenase. Thus the loss of binding of NADH to protein prevents the transport of electrons which in return reduces ATP production in the parasite. UV spectrophotometric result was a supporting observation to spectrofluorimetry.The difference between the excitation maxima in spectrofluorimetry and absorbance maxima in spectrophotometry in the present study might be due to different excitation wavelengths. S. alata exhibited a reduced fluorescence peak compared to control indicating that Senna extracts altered the membrane potential of parasite mitochondria.

Citrate synthase was used as a quantitative enzyme marker for detecting the presence and intergrity of intact mitochondria (Lanza and Nair 2009). Formation of coenzyme A from acetyl coenzyme A and oxaloacetate, by citrate synthase, is thus a key enzyme for driving the mechanism of oxidative ATP generation (Zinsser et al. 2013). The activity of citrate synthase decreased significantly when the cell or tissue underwent stress during aging as was observed in humans (Short et al. 2005). This observation was also found in the present study in all the plant treated worms suggesting inhibition of the enzyme activity, and at the same time might uncouple the mitochondria (Zinsser et al. 2013).

Mitochondrial enzyme were detected through native gel staining using specific substrates for each of them. The bands that were obtained indicated the expression of enzyme in both control and treated parasites but its activity through spectrophotometric studies showed inhibition. Similar observations were reported in helminths treated with quinazoline, bithinol, nafuredin and valproic acid (Ikuma et al. 1993; Omura et al. 2001; Matsumoto et al. 2008; Berger et al. 2010). Decrease in the activity of NADH fumarate reductase could have an effect on the reduction of fumarate to succinate and subsequently paralyse the anaerobic pathway reaction. Reduction of specific activity of succinate dehydrogenase and succinate quinone reductase in treated parasites may cause inhibition of the oxidation of succinate to fumarate in complex II and the transfer of reducing equivalents to ubiquinones, and might prevent the aerobic respiration.The level of succinate was not affected by rotenone which otherwise block the complex I activity in aerobically respiring cells but cannot inhibit succinate which is oxidized by complex II and has been reported to recover mitochondrial function in septic skeletal muscle (Protti et al. 2007; Karlsson et al. 2016). Reduction of specific activity of succinate dehydrogenase and succinate quinone reductase in treated parasites in the present study might cause inhibition of the oxidation of succinate to fumarate in complex II and the transfer of reducing equivalents to ubiquinones, thus could prevent aerobic respiration. Inhibition of the NADH oxidase might inhibit the conversion of reduced NADH to its oxidized form that remains as co-factors to various enzymes. The oxidation of reduced equivalents is a must for these oxidized cofactors to reunite with their enzyme counterparts. NADH oxidase play an essential role in aerobic metabolism and facilitates consumption of oxygen as the terminal electron acceptor in gram positive bacteria (Lucey and Condon 1986). Decrease in enyme activities is an indication to the stimulation of mitochondrial apoptotic factors and release of cytochrome C. Amongst the three plants S. alata showed more effct on the parasite, thus further study needs to analyse the phytochemical compound of this plant and its mechanism of action on the enzyme activity before claiming its potent anthelmintic activity.

Conclusions

The three species of Senna caused severe damage to the mitochondrial membrane, deformation of the cristae and consistency of the matrix. Inhibition of citrate synthase might decrease the functional integrity of mitochondria. The reduction in the activities of mitochondrial enzymes suggested invariable hindrance in the flow of electrons across the membrane and matrix to the final electron acceptor for both aerobic and anaerobic energy production. Loss of mitochondrial integrity resulted in loss of membrane potential. The decreased activity of the various enzymes might therefore cause improper biogenetic function of the mitochondria and eventually might have led to cell death in the parasite.

Supplementary Information

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Funding

The work was funded by UGC Single Girl Child Fellowship under Basic Scientific Research Fellowships provided to the first author (grant no. F.No. 25–1/2014–15 (BSR)/5–132/2007/(BSR)).

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.