INTRODUCTION

The order Santalales consists of 10 families and ca. 2000 species. The largest family is Loranthaceae (900), followed by the Santalaceae (400), Viscaceae (300), and Olacaceae (250). None of the six remaining families has more than a hundred species (^{Cronquist,1988}). According to the system proposed by the Angiosperm Phylogeny Group, the family Loranthaceae is classified with Olacaceae, Balanophoraceae, and Santalaceae, and others families in the order Santalales, Core Superasterids (^{APG IV, 2016}).

Loranthaceae is pantropical, with ca. 65 genera and 900 species of epiphytic, photosynthetic and hemiparasites plants that adhere to branchs of trees by means of haustoria to absorb water and nutrients. Hemiparasitic plants are commonly known as mistletoe (^{Lorenzi, 2000}; ^{Pennington et al., 2004}) or 'suelda con suelda' (Peruvian vernacular name). There are 11 genera and ca. 60 species in Peru. The only arborescent genus is Gaiadendron G., which is a root parasite (Pennington et al., 2004). Psittacanthus linearis (Killip) J.F. Macbr. attached to following forest species of the seasonally dry forest: Prosopis pallida (Willd.) Kunth (Fabaceae) (algarrobo), Acacia macracantha Willd. (Fabaceae) (faique), Salix humboltiana Willd. (Salicaceae) (sauce) and others species.

A parasitic plant is an angiosperm (flowering plant) that directly attaches to another plant via haustorium, which is a specialized structure that forms a morphological and physiological link between the parasite and host (^{Nickrent & Musselman, 2004}; ^{Yoshida et al., 2016}). Hemiparasitic plants have an ambiguous relationship with their hosts, which on the one hand, are the sources of inorganic nutrients but, on the other hand, can compete with the hemiparasites for light. Consequently, hemiparasitic plants have a unique way of acquiring resource that combines parasitism of other species with their own photosynthetic activity, so that despite their active photoassimilation and green habit, they acquire substantial amount of carbon from their hosts (Tĕšitel et al., 2009). It was investigated a model of the spatial distribution of true mistletoe, (Cladocolea loniceroides (Tiegh.) Kujit, using classical statistics, spatial statistics and geostatistics in the green areas of Tlalpan Delegation - Mexico City to analyze the correlation among mistletoe-hosts (^{Espinoza-Zúñiga et al., 2019}).

A study of the hemiparasitic angiosperm Thesium humile Vahl (Santalaceae) assessed physiological changes in the root before and after the attachment to the host plant (Triticum vulgare). This obliged angiosperm root hemiparasite can live in an autotrophic state for several weeks before joining the host. T. humile is able to take up water and nutrients ions from the soil, but has very high levels of Na and low levels of P (^{Fer et al., 1994}). The functional relationships between aerial and root parasitic plants and their woody hosts and consequences for ecosystems were recently discussed, and gross comparisons of nutrient content between infected and uninfected hosts, or parts of hosts, have been widely used to infer basic differences or similarities between host and parasites (^{Bell & Adams, 2011}). A study of the hemiparasite Santalum album L. (Santalaceae) and its hosts in southern China compared two non-N2-fixing hosts [Bischofia polycarpa (H. Lév.) Airy Shaw (Phyllanthaceae) and Dracontomelon duperreanum Pierre (Anacardiaceae)] and two N2-fixing hosts (Acacia confusa Merr. and Dalbergia odorifera T.C. Chen, both species within Fabaceae) with respect to the growth characteristics and nitrogen nutrition of S. album (^{Lu et al., 2014}).

Studies about in vitro tissue culture, phytochemical analysis and antibacterial activity are scarce in the species of Loran-thaceae, and in the case of P. linearis are non-existent.

^{Deeks et al. (1999)} reviewed in vitro tissue culture of parasitic flowering plants in genera of the Loranthaceae family such as Amyema Tiegh., Amylotheca Tiegh., Dendrophthoe Mart., Nuytsia R. Br. ex G. Don, Scurrula L., Tapinanthus (Blume) Rchb, and Taxillus Tiegh., and discussed the applications of tissue culture techniques in studies of the biology and host-pathogen interactions.

Callus induction, seedlings (with haustorial discs, holdfasts and plumular leaves), embryogenic callus, shoots, and somatic embryos, were the main results obtained (cf. ^{Deeks et al., 1999}). Relevant results have been reached for Dendrophthoe falcata (L. f.) Ettingsh, one of the most studied species (^{Ram et al., 1993}), and for Amyema miquelii (Lehm. ex Miq.) Tiegh., A. quandang (Lindl.) Tiegh., and A. pendula (Sieber ex Spreng.) Thieg., where callus was induced, and the seedlings formation with several structures was obtained (^{Hall et al., 1987}).

Several species of Loranthaceae are of ethnobotanical importance and are used as medicinal plants in various regions of the world, especially in Africa and India. African mistletoes of the Loranthaceae (Globimetula Tiegh., Phragmanthera Tiegh., Agelanthus Tiegh. and Tapinanthus, and Viscaceae family as Viscum L.) are hemiparasitic plants and their preparations in the form of injectable extracts, infusions, tinctures, fluid extracts or tea bags are widely used in various cultures and in almost every continent to treat or manage health problems including hypertension, diabetes mellitus, inflammatory conditions, irregular menstruations, menopause, epilepsy, arthritis, and cancer (^{Adesina et al., 2013}). A preliminary phytochemical screening of the methanolic extract of Helicanthes elastica (Desr.) Danser (Loranthaceae) which grows on the host plants Nerium indicum Mill. (= N. oleander L.) (Apocynaceae) and Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. (Euphorbiaceae) revealed the occurrence of various constituents such as glycosides, saponins, tannins and phenols in both hosts (^{Kumar & Mathew, 2014}). On the other hand, a in vitro study about the anti-diabetic properties from hemiparasitic species of D. falcata revealed that its plant's leaves extracts had inhibitory activity on the key enzyme alpha-amylase, which enzyme breaks the large strach molecules that produces free glucose and simultaneously increases the blood sugar level, and consequently hyperglycemia (^{Naskar et al., 2019}). Likewise, the cloroform fraction and crude extract from Loranthus acaciae Zucc grown in Saudi Arabia showed anti-diabetic, anti-inflammatory and antioxidant activities (^{Noman et al., 2019}). Leaves from Scurrula parasitica L., quercetin, quercitrin, kaempferol 3-O-a-L-rhamnoside, (+)-catechin compounds, together with ethyl acetate and methanol extracts exhibited effective antioxidant activities against DPPH(2,2-diphenyl-1-picrylhydrazyl), ABTS[2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] and FRAP (Ferric reducing antioxidant potential), while n-hexane and other compounds were inactive (^{Muhammad et al., 2019}).

Other preliminary phytochemical, physicochemical, and antimicrobial studies of Loranthus elasticus Desv. [= Heli-canthes elasticus (Desv.) Danser] associated to the neem tree (Azadirachta indica A. Juss.) (Meliaceae) and collected from the local fields of Salem district of Tamil Nadu, India, were performed (^{Krishnaveni et al., 2016}). An eth-nobotany study of seven hemiparasitic Loranthaceae species: Tapinanthus bangwensis (Engl. & K. Krause) Danser, T. belvisii (DC.) Danser, T. sessilifolius var. glaber Balle (= Tapinanthus praetexta Polhill & Wiens), Phragmanthera capitata (Spreng.) Balle, P. capitata var. alba, Globimetula braunii (Engl.) Danser and G. dinklagei subsp. assiana Balle [= Globimetula assiana (Balle) Wiens & Polhill], all of which are used in traditional medicine in the Sud-Comoé region (Côte d'Ivoire) (Amon et al., 201 7). Likewise, an ethnobotanical analysis of parasitic plants (Parijibi) in the Nepal Himalaya, a hotspot of botanical diversity, housing 15 families and 29 genera of plants with parasitic or mycoheterotrophic habit (^{O'Neill & Rana, 2016}).

On the other hand, in miscellaneous studies, African mistletoe [Tapinanthus dodoneifolius (DC.) Danser] [= Agelanthus dodoneifolius (DC.) Polhill & Wiens] commonly called 'Kauchi' in Hausland (Northern Nigeria) is currently used ethnomedicinally in the inhibition of the growth of Agrobacterium tumefaciens, Escherichia coli, Salmonella sp., Proteus sp., and Pseudomonas sp., species of bacteria associated with gastrointestinal tract and wound infections (^{Deeni & Sadiq, 2002}). The thin layer chromatography (TLC) phytochemical screening in some Nigerian Loranthaceae as Phragmanthera, Globimetula and Tapinanthus genus, collected from the field was carried out with a view to ascertaining chemical constituents present and determining their importance in the taxonomic delimitation of the taxa (^{Wahab et al., 2010}). In another study in Phragmanthera capitata 11 endophytic fungi species from four genera: Aspergillus (six species), Penicillium (three species), Trichoderma (one species) and Fusarium (one species). In the same study the phyto-chemical analysis revealed the presence of flavonoids, anthroquinones, tannins, phenols, steroids, coumarins and terpenoids, and absence of alkaloids and saponins in all the extracts (^{Ladoh-Yemeda et al., 2015}). Micromorphological studies of the Loranthaceae P. capitata with scanning electron, light, and energy dispersive X-ray (EDX) microscopies revealed a paracytic type of stomata, oval-shaped lenticels, densely packed stellate trichomes, tracheary elements, which are tightly packed with granules believed to be proteins, and deposits chiefly composed of Si, Al, K, and Fe (^{Ohikhena et al., 2017}). Likewise, qualitative and quantitative phytochemistry, micro and macro-elements and microscopy of Tapinanthus dodoneifolius (African mistletoes) collected from 'guava' (Psidium guajava L.) (Myrtaceae), 'rubber' (Hevea brasiliensis), and 'orange' [Citrus sinensis (L.) Osbeck] (Rutaceae) trees was carried out with the purpose of comparing their pharmacological and biological contents. The phytochemical analysis of African mistletoes showed the presence of oxalate, phytate, saponin, alkaloid, glycoside, and tannin, which were present in proportions that varied with their hosts (^{Idu et al., 2016}).

The aim of the present study was to evaluate some aspects of in vitro tissue culture, preliminar phytochemical analysis and antibacterial activity of Psittacanthus linearis collected from the host Prosopis pallida, a representative species of the Seasonally Dry Forest of Northern Peru, due to its high tolerance to drought and salinity and produce fruits with high nutritional value.

MATERIALS AND METHODS

Plant materials and seed desinfection. Seeds and whole plants with red flowers of [Psittacanthus linearis (Killip) J.K. Macbride] 'suelda con suelda'(Loranthaceae), in Prosopis pallida 'algarrobo' (Figure 1a) were collected from a natural population in Pítipo district of Ferreñafe province, Lambayeque-Peru. The plant specimens were identified by Dr. Santos Llatas Quiroz and botanist José Ayasta Varona from the PRG Herbarium of the Faculty of Biological Sciences of the Universidad Nacional Pedro Ruiz Gallo (Lambayeque, Peru). Specimens were deposited in the Herbarium PRG with the voucher number 18013. The seeds were manually dehusked and surface sterilized by immersion for 1 min in ethanol 70% (v/v) and 5 min in sodium hypochlorite solution (Clorox^®) 5.25% (v/v) containing a few drops of polyoxyethylene sorbitan monolaurate (Tween 20^®), followed by five rinses of 1 min each with sterile distilled water.

Figure 1 Plants and various process in in vitro tissue culture of Psittacanthus linearis, a. Adult plant of P. linearis ('suelda con suelda') on host Prosopis pallida ('algarrobo'), b. Germinated seeds with holdfast formation and green and compact callus, in culture medium supplemented with 2.0 mg L¹ BAP, c. Seeds of P. linearis adhered to branches of the host, observing some growing seedlings, d. Green callus formed in the holdfast, in culture medium supplemented with 2.0 mg L"¹ BAP, showing organogenic primordia, e. Seedling obtained from seed, propagated in culture medium supplemented with 2.0 mg L¹ BAP, after 120 days of culture. 

Culture media and culture conditions. All media consisted of full-strenght MS (^{Murashige & Skoog, 1962}) salt formulation containing the following ingredients: 1.0 mg L^-1 thiamine.HCl, 100 mg L^-1 myo-inositol, 3% sucrose, and 0.6% agar-agar. The disinfected seeds were germinated aseptically in the MS formulation supplemented with 3-indol acetic acid (IAA) and 6-benzilaminopurine (BAP). The seeds were scored daily for germination and the breakthrough of the radicle from the seed coat were used as the criterion for germination (^{Côme, 1968}). In the leaves and apical buds elongation, BAP and IAA-GA3 (Gibberellic acid) combinations were applied in several concentrations. In the callus induction, three types of auxins [2,4-D (2,4-dichlorophenoxyacetic acid), NAA (naphthalene acetic acid), and IAA] were applied in several concentrations, and for organogenesis, IAA-BAP were also applied in several concentrations. The pH of all the culture media was adjusted to 5.7±0.1 with KOH or HCl before autoclaving. For all experiments, 15 mL of the medium was transferred to 150x25 mm test tubes, covered with polypropylene tops, and autoclaved for 20 min at 121 ^oC and 1.05 kg cm^-2. One explant was cultured per tube. Cultures were incubated at 26±2 ^oC under a 16-h photoperiod with the light intensity of 70 Mmol m^-2 s^-1 photosynthetic active radiation provided by cool white fluorescent tubes; only the callus cultures were incubated in a dark room.

Phytochemical analysis. Stems, leaves, and fruits of P. linearis were oven-dried separately at 40 ^oC and powdered (5 to 10 mm of particle diameter). The dried powder was subjected to extraction with 150 mL of 96^o ethanol (three extractions of 50 mL) during 24 h. All extracts were evaporated to dryness under reduced pressure. The extract was recovered with ethanol and the remnant with chloroform to form the ethanolic and chloroformic fractions. Subsequent analysis included colorimetric quantification of total polyphenols, determination of proteins and Total Radical-Trapping Antioxidant Parameter (TRAP) (^{Lissi et al., 1992}, ^{Wagner et al., 1998}), and characterization of flavonoids by means of two-dimensional chromatography.

Calorimetrie quantification of total polyphenols. Samples (stems, leaves and fruis) of 1 g air-dried material were extracted with MeOH (50:50) for 48 h and dark conditions. For this process 3 mL ferric chloride and 3 mL potassium ferrocyanide were used, and the readings were made in photocolorimeter with an absorbance of 720 nm. The standard curves were established with tannic acid.

Detection of proteins by Sodium Dodecyl sulfate Poly-acrylamide gel Electrophoresis (SDS-PAGE). SDS-PAGE was performed according to the method of ^{Laemmli (1970)} using a discontinuous system of two layers: 10% polyacrylamide resolving gel and 4% polyacrylamide stackin gel. Samples (leaves) of 1 g air-dried material were extracted with 10 mL of 20 mM Tris-HCl buffer pH 8.0, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM β mercaptoethanol (ßME) and 1% polyvinylpyrrolidone (PVP). Electrophoresis was carried out at 150 mA/gel for 45 min using a Mini Protean II Electrophoresis System (Bio Rad, USA), and gels were stained with silver. Molecular weight standars SDS-VII (Sigma Chem. Co., St Louis MO) were used (^{Wagner et al., 1998}).

Determination of Total Reactive Antioxidant Potential (TRAP). Total Reactive Antioxidant Potential (TRAP) was measured by luminol-enhanced chemilumininescence (^{Lissi et al., 1992}). The reaction medium consisted of phosphate buffer 100 mM (pH 7.4), 20 mM 2,2'-azo-bis(2 -amidinopropane) (ABAP), 10 μΜ luminol in 0.1 M NaOH. Incubation of the mixture at room temperature generates a nearly constant light intensity that was measured directly in a Packard tri-Carb scintillation counter with the circuite coincidence out of mode. The system was calibrated with catechin, quercetin and ascorbic acid.

Caracterization of flavonoids. For this analysis, 5.0 mg samples (stems and leaves) of methanolic extracts from P. linearis were disolved in 10 mL MeOH (80:20). They were purified or fractionated by column chromatography and analyzed by TLC and UV spectroscopy (^{Mabry et al., 1970}; ^{Waterman & Mole, 1994}).

We also compared protein profiles of leaves of P. linearis (PH) and Ligaria cuneifolia from different hosts 20, 24, 30 and 3. L. cuneifolia has been studied with respect to its anatomical, phytochemical and immunological properties (^{Wagner et al., 1998}), and in Argentina leaf and stems infusions have been used as a substitute of Viscum album based on their putative depressive effect on high blood pressure.

Antibacterial activity. Three strains of Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli and Pseudomonas aeruginosa) bacteria, were isolated from clinical samples from the Microbiology Laboratory (Faculty of Biological Sciences of the Universidad Nacional Pedro Ruiz Gallo, Lambayeque, Peru) and identified by microscopic observation and biochemical tests. In the dilutions and inoculum preparations, the ethanolic and chlorophormic fractions of wild plants were weighed and disolved in sterile distilled water to prepare the required dilutions of 0.0 (control), 50, 100, 200 and 300 Mg mL^-1 per disc. Inocula of bacterial species were prepared in nutrient medium Müller Hinton and kept under incubation at 37 ^oC for 8 h for sterilization control. The cultures were then refrigerated at 2-8 ^oC. For the Kirby-Bauer Disc Diffusion Test, 1 mL of inoculum suspension was spread uniformily over the agar medium using a sterile glass rod to get uniform distribution of bacteria. Sterile discs of 5 mm diameter prepared with Whatman paper filter N^o 41 were impregned with different concentrations of 0.0, 50, 100, 200 and 300 Mg mL^-1 per disc of plant extract. The discs were placed on the medium and incubated at 37 ^oC for 24 h. Antibacterial activity was determined by measuring the width of the inhibition zone (mm). Equivalent concentrations to the tube N^o 5 McFarland nephelometer which corresponding to a density of 1.5x10⁸ ufc mL^-1, were used.

Statistical analysis. The data (means ± SE, n = 10) were subjected to one-way analysis of variance (ANOVA) and the Tukey HSD multiple range test P ≤ 0.05 level, in order to compare treatment means. All analyses were performed with SPSS version 11.5 (SPSS Inc., Chicago, IL, USA).

RESULTS

In vitro tissue culture

In vitro seed germination. Data of the P. linearis seed evaluation are presented in the table 1. The contamination rate in some treatments was of 10%, and Aspergillus and Cladospora were observed; the browning was observed between 10 to 20%, in all the treatments tested. Cotyledons emerged in 7 days, and complete expansión of cotyledons occured between 50 and 60 days. After 60 days, 100% of seeds treated with 2.0 mg L^-1 BAP germinated and reached the maximum leaf elongation (5.2 mm) and the largest diameter of the holdfast (6.7 mm) after 90 days, the elongation of the leaves was 12.3 mm (Figure 1b). In other experiment, 100% of seeds adhered to host branches Acacia macracantha (faique) germinated after 7 days (Figure 1c).

Table 1  In vitro seeds germination of Psittacanthus linearis after 60 days of culturea. 

^aMeans followed by the same letters do not significantly differ according to a Tukey test at a 5% probability.

^bCont., Fungal contamination; ^cBrown., browning; ^dSeed germ., seed germination

^e1-2 leaves; 3-4 leaves; 5 to > leaves

Leaves elongation. The highest leaves elongation (8.8 mm) was observed in MS culture medium supplemented with 0.02 mg L^-1 IAA and 0.02 mg L^-1 GA₃, with an average (40%) of seedlings with 5-7 leaves per explant (germinated seed more holdfast). We also observed a significant formation of green and compact callus (70%), with organogenic appearance (Table 2), after 60 days of culture (Figure 1d).

Table 2 Size and number of leaves in the holdfast of Psittacanthus linearis after 60 days of culture. 

"Means followed by the same letters do not significantly differ according to a Tukey test at a 5% probability.

Callus induction and organogenesis. Callus induction (90%) in physiological condition (+++) was observed in MS culture medium supplemented with 2.0 mg L^-1 BAP, evaluated over 150 days of culture; likewise, 10-25% of organogenic callus (indirect organogenesis) showed structures similar to apical buds (Table 3, figure 1e). Other formulations of culture medium - IAA (0.5, 1.0 and 2.0 mg L^-1), NAA (0.5, 1.0 and 2.0 mg L^-1) and 2,4-D (0.125, 0.25 and 0.5 mg L^-1) - showed between 20-40% green and compact callus formation.

Table 3 Callus induction and indirect organogenesis in the holdfast of Psittacanthus linearis after 150 days of culture. 

^a-, without callus formation; +, callus < 5 mm ø; ++, callus between 6-10 mm 0; +++, callus > 10 mm ø. ^aMeans followed by the same letters do not significantly differ according to a Tukey test at a 5% probability.

Phytochemical analysis

In the determination of total polyphenols, employing tannic acid as a standard, we observed a higher concentration in red flowers (171.51 mg/dry mass) and a lower concentration in leaves (36.27 mg/dry mass); in stems and fruits the concentración of tanic acid was 42.62 and 146.70 mg/dry mass, respectively. In the characterization of flavonoids, two glycosidated (kaempherol glicoside and quercetin glicoside) and two non-glycosidated (kaempherol and quercetin) were found. The color reactions indicated the presence of free and glycosidated quercetin and free and glycosidated kaemferol (Figure 2a). In the amylic fraction of the proanthocyanidins analysis where was used the acid treatment of the methanolic extract of stems and leaves, was detected cyanidin, where the procyanidin (monomer catechin) by acid treatment is transformed into cyanidin (Figure 2b). Likewise, in a comparative study on the detection of proteins in leaves of P. linearis and leaves of Ligaria cuneifolia (R. et P.) Tiegh., a species already studied in our laboratory, from different hosts of L. cuneifolia (20, 24, 30 and 31) was observed that the band 22.6 kD presents in P. linear-is is absent in specimens of L. cuneifolia (Figure 2c).

Figure 2 Structure of some chemical compounds detected in Psittacanthus linearis and antibacterial activity of the ethanol extract on strains S1 and S2 of S. aureus a. Chemical structuren of flavonoids quercetin and kampferol, b. Chemical structure of the catechin that by acid treatment originates cyanidin, c. Comparative protein profile between leaves of P. linearis (PH) and leaves of Ligaria cuneifolia from different hosts 20, 24, 30 y 31 and d. Antibacterial activity of the ethanolic extract of stems on strains S1 and S2 of S. aureus. 

Antibacterial activity

Among the three bacterial species tested, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, each with three strains, respectively, only strains of S. aureus showed sensitivity to the extracts of P. linearis. Additionally, the ethanolic extracts were found to be more active than the chloroformic extracts. Fruit extracts were more active than stem and leaves extracts. Concentrations of 300 Mg mL^-1 were more active than concentrations of 200 and 100 Mg mL^-1, and strain S1 of S. aureus was more sensitive than strains S2 and S3 (Table 5). The highest inhibition halo (16.7 mm) was observed in the ethanolic extract of fruits at concentrations of 100, 200 and 300 Mg mL^-1 when tested against strain S1 (Table 5, figure 2d).

Table 4 Qualitative phytochemical analysis of several plant samples of Psittacanthus linearis in Prosopis pallida. 

BAA: η butane: acetic acid: water (4:1:5. upper phase CAA: chloroform: acetic acid (2:1, saturated with water) Forestal: hydrochloric acid (CCI: acetic acid: water (3:30:10) Rf: Distance covered by the standard/versus the soKent Formic: formic acid: acetic acid: water (9:2:3)

Table 5 Antimicrobial activity of both chlorophormic and ethanolic extracts of various plant samples of Psittacanthus linearis against Staphyloccus aureus strains³. 

^aPo:it. Poiition: Bact Str.. Bacteria Strein: Conct ipg Di, Concentration ipg/Di;o: Inhib. Signif. Inhibition/Significance.

DISCUSSION

In the most 'mistletoes' of the Loranthaceae, a radicle emerges during seed germination which attaches and penetrates the host. The plumule (embryogenic shoot) emerges from between the two cotyledons (^{Bajaj, 1970}). This dependency of the parasite on host stimulus for seed germination and the chemical factors initiating haustorium formation were studied in tissue culture (^{Bhatnagar, 1987}).

Between the decades 70s to 90s, several classic studies using tissue culture were performed in various species of the Loranthaceae such as Amyema and Amylotheca, two genera of Australian mistletoes. ^{Hall et al. (1987)} studied the effects of hormone and nutrients on development and morphology of leaves, callus and seedlings grown on modified MS medium containing IAA or NAA in Amyema and found that seedlings formed plumular leaves and haustorial discs in vitro. Likewise, mature embryos from Amylotheca dictyophleba (F. Muell.) Tiegh. on White's medium, with casein hydrolysate and IAA, developed seedlings with holdfasts, haustorial disc, and plumular leaves (^{Bajaj, 1970}). D. falcata in White's medium developed undifferentiated and embriogenic callus embryoids, buds (shoot, floral), and seedlings with holdfasts and haustorial discs (^{Ram & Singh, 1991}). Nuytsia floribunda R. Br. in White's medium, with casein hydrolysate, IBA, and KIN, produced shoots, roots, embryogenic callus, and somatic embryos (^{Nag & Johri, 1969}). Scurrula pulverulenta (Wall.) G. Don mature endosperm on White's medium, with casein hydrolizate and IAA, produced callus, shoots, seedlings with haustoria, and somatic embryos (^{Bhojwani & Johri, 1970}). Tapinanthus bangwensis (Engl. & K. Krause) Danser, on White's medium produced callus, shoots, and seedlings with embryo germination and holdfast formation occurring without a host (^{Onofeghara, 1972}). In both, Taxillus vestitus (Wall.) Danser and T. cuneatus (Heyne) Danser several factors that affecting organogenesis were studied and it was also founded that the development of shoot buds and haustoria was influenced by growth regulator combinations (^{Johri & Nag, 1970}; ^{Nag & Johri, 1976}). In all these studies, morphogenic processes were initiated with seed germination and holfast induction and continued with callus formation and elongation of the cotyledons, as was observed in the present study with P. linearis, where only callus formation and elongation of cotyledonal leaves were the most relevant results. In another parasitic species, western hemlock dwarf mistletoe [Arceuthobium tsugense (Rosend.) G.N. Jones], the most evolutionarily specialized genus of Santalaceae (ex Viscaceae), seedlings developed from split radicles and split holdfasts after 5 months in culture (^{Deeks et al., 1997}). In the present study a similar or longer period of time was required to induce holdfast with green callus for the seedlings of P. ligularis.

Phytochemical constituents of the leaves of African mistletoe (Tapinanthus dodoneifolius), an ethnomedicinal plant of Northern Nigeria, obtained from 14 different hosts, showed the common occurrence of anthraquinones, saponins, tannins, a rare presence of alkaloids and the absence of phlobatannins in the hemi-parasite (^{Deeni & Sadiq, 2002}). Likewise, preliminary phytochemical screening of the methanolic extracts of H. elastica in hosts, N. indicum and H. brasiliensis, revealed the occurrence of various constituents as alkaloids, glycosides, flavonoids, phenols, tannins, sterols, triterpenoids, diterpenes and carbohydrates, but not proteins or amino acids (^{Kumar & Mathew, 2014}). In another study, phytochemical analysis of endophytic fungi from stems of Phragmanthera capitata, harvested in Theobroma cacao L., revealed the presence of flavonoids, anthroquinones, tannins, phenols, steroids, coumarins and terpenoids, but absence of alkaloids and saponins, in all acetate ethyl extracts (^{Ladoh-Yemeda et al., 2015}). Phytochemical constituents from L. elasticus presented in the Neem Tree (A. indica), namely alkaloids, amino acids, anthocyanin, carbohydrates, cardiac glycosides, coumarins, diterpenes, emodins, fatty acids, phlobatannin, phenols, saponin and terpenoids, were presented in methanolyc extract, whereas the flavonoids, glycosides, leucoanthocyanin, phytosterol, proteins, steroids and tannin were absent in methanol extract. Small amounts of saponins were noticed in alcohol and water extracts, and in water extracts of the leaves shows moderate amounts of gums and mucilage (^{Krishnaveni et al., 2016}). The phytochemical analysis of L acaciae led to the isolation and characterization of quercetin 3-O-β-D-glucopyranoside, quercetin 3-O- β -(6-O-galloyl)-glucopyranoside, (-) catechin, and catechin 7-O -gallate. Among these compounds quercetin 3-O- β -D-glucopyranoside, quercetin 3-O- β -(6-O-galloyl)-glucopyranoside and catechin 7-O-gallate, were isolated for the first time from this species (^{Noman et al., 2019}). In Plicosepalus curviflorus (Oliv.) Tiegh., a semiparasitic plant grown in Saudi Arabia, traditionally used as a cure for diabetes and cancer in human, a flavonoid quercetin, (-)-catechin, and a flavane gallate 2S,3R-3,3',4',5,7-pentahydroxyflavane-5-O-gallate, were isolated from the aerial parts (^{Orfali et al., 2019}). Cold extractions of S. parasitica leaves were carried out using n-hexane, ethyl acetate and metanol to obtain the crude extracts, and purification of the extracts led to isolation of quercetin, quercitrin, kaempferol 3-O-α-L-rhamnoside, (+)-catechin, lupeol 5, lupeol palmitate, ß-sitosterol, squalene, octaco-sane, octadecane and eicosane (^{Muhammad et al., 2019}). Only polyphenols and four flavonoids, two glycosidated (kaempherol glicoside and quercetin glicoside) and two non-glycosidated (kaempherol and quercetin), were detected in the study carried out on P. linearis, without deepening in the isolation and identification of other secondary metabolites.

In the study on the relationship between mistletoes-hosts, thirty field collections of specimens of the genus Phragmanthera, Tapinanthus and Globimetula (Loranthaceae), obtained from various localities of Nigeria, with several hosts plants being parasitized by the mistletoes, were phytochemical screened. Most of the samples were slightly positive for alkaloids, anthraqui-none-related compounds, terpenoids and terpenoid-related compounds; however, ketonic compounds were of rare ocurrence in all the samples (^{Wahab et al., 2010}). Analysis of Tapinanthus dodoneifolius (DC.) Danser, collected from P. guajava, H. brasiliensis and Citrus sinensis (L.) Osbeck showed the presence of oxalate, phytate, saponin, alkaloid, glycoside and tannin (^{Idu et al., 2016}). Certainly, not only is very necessary the study of the relation P. linearis with P. pallida (algarrobo) but also with other species of the seasonally dry forest such as A. macracantha (faique), Loxopterygium huasango Spruce ex Engl. (hualtaco), and others.

In all of these studies, the presence of phenols was common; flavonoids, proteins, and amino acids were only detected in some species, which indicates that the species of the Loranthaceae family present a broad spectrum of secondary metabolites. Likewise, the occurrence of different protein bands between the species of P. linearis and L. cuneifolia can be partially used in the differentiation of genera, as indicated in the study by ^{Wahab et al. (2010)} who also concluded that chemical characters of thirty field collections from various Loranthaceae species may only be used as supporting evidence in the identification and delimitation of the taxa. However, studies of proteins in Loranthaceae species are scarce. Proteins from L. elasticus were reported, but without specific identification (^{Krishnaveni et al., 2016}). In the trials on total antioxidant activity (TRAP), in leaf extract of P. linearis exhibited a decrease in chemiluminescence when was compared with L. cuneifolia (^{Wagner et al., 1998}). This phenomenon was for a period proportional to the amount of oxidants present in the sample, a process that occurred until the regeneration of the luminol radicals, expressing the results in mMol of catechin. In other species of Loranthaceae in Peru, these comparative methods can also be used for the differentiation of genera and species.

There are few studies about the antimicrobial activity exerted by extracts of Loranthacaeae species. The antimicrobial activity of L. elastica extracts showed that all the organisms are inactive in the organic solvents up to a level of 200 Mg mL^-1, and both Gram negative (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and Gram positive (Bacillus subtilis and Staphylococcus aureus) organisms show activity in the methanol and ethanol extracts at a concentration of 25 Mg mL^-1; the ethanol and methanol extracts show positive for all organisms tested (^{Krishnaveni et al., 2016}). In another study, screening of the Tapinanthus dodoneifolius, obtained from 14 different hots, revealed a wide spectrum of antimicrobial activities against drug-resistant bacteria such as Agrobacterium tumefaciens, Bacillus sp., Escherichia coli, Salmonella sp., Proteus sp. and Pseudomonas sp., all of which are known to be associated with either crown gall or gastrointestinal tract and wound infections (^{Deeni & Sadiq, 2002}). Likewise, all extracts and isolated compounds of S. parasitica leaves showed weak activity on antimicrobial activity against two Gram-positive bacteria (Staphylococcus aure-us and Bacillus subtilis), two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and a fungi (Aspergillus niger) with the exception of quercetin which exhibited moderate activity against P. aeruginosa with MIC and MBC value of 250 μg/mL (^{Muhammad et al., 2019}). In the study with P. linearis, although the results were only significant with strains of Staphylococcus aureus, its is necessary to carry out other tests with different strains of bacterial species of wide prevalence in hospital infections.

CONCLUSION

In this work, carried out in Psittacanthus linearis (suelda con suelda), a representative species of the BES of Lam-bayeque (Peru), we found that in vitro tissue culture allows germination of seeds, clonal propagation, callus induction and organogenesis. Likewise, some phytochemical aspects were studied and their biological activity on certain bacterial pathogens was demonstrated.